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1.
Adv Exp Med Biol ; 1354: 49-62, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34807436

RESUMO

The period of conceptus (embryo and extraembryonic membrane) development between fertilization and implantation in mammalian species is critical as it sets the stage for placental and fetal development. The trophectoderm and endoderm of pre-implantation ovine and porcine conceptuses undergo elongation, which requires rapid proliferation, migration, and morphological modification of the trophectoderm cells. These complex events occur in a hypoxic intrauterine environment and are supported through the transport of secretions from maternal endometrial glands to the conceptus required for the biochemical processes of cell proliferation, migration, and differentiation. The conceptus utilizes glucose provided by the mother to initiate metabolic pathways that provide energy and substrates for other metabolic pathways. Fructose, however, is in much greater abundance than glucose in amniotic and allantoic fluids, and fetal blood during pregnancy. Despite this, the role(s) of fructose is largely unknown even though a switch to fructosedriven metabolism in subterranean rodents and some cancers are key to their adaptation to hypoxic environments.


Assuntos
Embrião de Mamíferos , Placenta , Animais , Implantação do Embrião , Endométrio , Feminino , Frutose , Gravidez , Ovinos , Suínos , Útero
2.
Adv Exp Med Biol ; 1354: 109-125, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34807439

RESUMO

Water transport during pregnancy is essential for maintaining normal growth and development of conceptuses (embryo/fetus and associated membranes). Aquaporins (AQPs) are a family of small integral plasma membrane proteins that primarily transport water across the plasma membrane. At least 11 isoforms of AQPs (AQPs 1-9, 11, and 12) are differentially expressed in the mammalian placenta (amnion, allantois, and chorion), and organs (kidney, lung, brain, heart, and skin) of embryos/fetuses during prenatal development. Available evidence suggests that the presence of AQPs in the conceptus mediates water movement across the placenta to support the placentation, the homeostasis of amniotic and allantoic fluid volumes, as well as embryonic and fetal survival, growth and development. Abundances of AQPs in the conceptus can be modulated by nutritional status and physiological factors affecting the pregnant female. Here, we summarize the effects of maternal dietary factors (such as intakes of protein, arginine, lipids, all-trans retinoic acid, copper, zinc, and mercury) on the expression of AQPs in the conceptus. We also discuss the physiological changes in hormones (e.g., progesterone and estrogen), oxygen supply, nitric oxide, pH, and osmotic pressure associated with the regulation of fluid exchange between mother and fetus. These findings may help to improve the survival, growth, and development of embryo/fetus in livestock species and other mammals (including humans).


Assuntos
Aquaporinas , Membranas Extraembrionárias , Âmnio/metabolismo , Animais , Aquaporinas/genética , Embrião de Mamíferos , Membranas Extraembrionárias/metabolismo , Feminino , Humanos , Placenta/metabolismo , Gravidez , Água/metabolismo
3.
Theriogenology ; 178: 30-39, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34775199

RESUMO

The use of different sires influences in vitro embryo production (IVP) outcome. Paternal effects are observed from the first cleavages until after embryonic genome activation (EGA). Little is known about the mechanisms that promote in vitro fertility differences, even less about the consequences on embryo development. Therefore, this study aimed to evaluate the paternal effect at fertilization, embryo developmental kinetics, gene expression and quality from high and low in vitro fertility bulls. A retrospective analysis for bull selection was performed using the In vitro Brazil company database from 2012 to 2015. The dataset was edited employing cleavage and blastocyst rates ranking a total of 140 bulls. Subsequently, the dataset was restricted by embryo development rate (blastocyst/cleaved rate) and ten bulls were selected as high (HF; n = 5) and low (LF; n = 5) in vitro fertility groups. IVP embryos derived from high and low fertility bulls were classified according to their stage of development (2 cells, 3-4 cells, 6 cells, 8-16 cells), at 24, 36, 48, 60, 72 hpi, respectively, to evaluate embryo kinetics. Pronuclei formation (24 hpi), cleavage rate (Day 3), development rate, and blastocyst morphology (Grade I and II - Day 7) were also assessed, as well as the abundance of 96 transcripts at 8-16 cell stage and blastocysts. There was no difference in early embryo kinetics (P > 0.05), and cleavage rate (HF = 86.7%; LF = 84.9%; P = 0.25). Nevertheless, the fertilization rate was higher on HF (72%) than LF (62%) and the polyspermy rate was lower on HF compared to LF (HF:16.2% LF:29.2%). As expected, blastocyst rate (HF = 29.4%; LF = 16.0%; P < 0.0001) and development rate (HF = 33.9% LF = 18.9%; P < 0.0001) were higher in HF than LF. At the 8-16 cell stage, 22 transcripts were differentially represented (P ≤ 0.05) between the two groups. Only PGK1 and TFAM levels were higher in HF while transcripts related to stress (6/22, ∼27%), cell proliferation (6/22, ∼27%), lipid metabolism genes (5/22, ∼23%), and other cellular functions (5/22, ∼23%) were higher on LF embryos. Blastocysts had 9 differentially represented transcripts (P ≤ 0.05); being only ACSL3 and ELOV1 higher in the HF group. Lipid metabolism genes (3/9, 33%) and other cellular functions (6/9, 67%) were higher in the LF group. In conclusion, the timing of the first cleavages is not affected by in vitro bull fertility. However, low in vitro fertility bulls presented higher polyspermy rates and produced 8-16 cells embryos with higher levels of transcripts related to apoptosis and cell damage pathways compared to high in vitro fertility ones. Evidence such as polyspermy and increase in apoptotic and oxidative stress genes at the EGA stage suggest that embryo development is impaired in the LF group leading to the reduction of blastocyst rate.


Assuntos
Fertilização In Vitro , Herança Paterna , Animais , Blastocisto , Bovinos , Embrião de Mamíferos , Desenvolvimento Embrionário , Fertilização In Vitro/veterinária , Masculino , Estudos Retrospectivos
4.
Methods Mol Biol ; 2303: 579-593, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34626408

RESUMO

Cell surface-tethered heparan sulfate glycosaminoglycan chains primarily function in a cell autonomous manner, while extracellular matrix-associated heparan sulfate glycosaminoglycan chains function in a non-cell autonomous manner. In addition, the cleaved forms of cell surface-tethered heparan sulfate chains enzymatically released by proteases and heparanases, called shedding, can contribute to non-cell autonomous mechanisms. The movement of heparan sulfate chains to surrounding cells mediated by transcytosis or filopodia also involves another non-cell autonomous mechanism. To determine cell autonomous or non-cell autonomous roles of heparan sulfate glycosaminoglycan chains during early embryogenesis, direct conclusions can be drawn by analyzing chimeric embryos which are composed of wild-type and heparan sulfate glycosaminoglycan chain-deficient cells. Here, we describe methods of production of these chimeric embryos and analysis of their cellular phenotypes with immunohistochemistry at a single-cell level.


Assuntos
Glicosaminoglicanos/química , Animais , Membrana Celular , Embrião de Mamíferos , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato , Camundongos
5.
Theriogenology ; 178: 8-17, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34735978

RESUMO

Successful implantation of porcine conceptus requires synergistic interaction with various signal molecules in the maternal uterus. Extracellular vesicles (EVs) in uterine luminal fluid (ULF) of mice play important roles in conceptus development. However, studies have not explored the roles of extracellular vesicles (EV) in ULF of pigs. The aim of this study was to identify characteristics, origin, and roles of ULF-derived EVs on day 9 of the estrous cycle and on day 9,12 and 15 of pregnancy in pigs. Western blot, BCA assay and HE staining analysis showed increase in EVs concentration in ULF began from day 12 of pregnancy. Immunofluorescence staining and transmission electron microscopy analysis showed that EVs were mainly derived from endometrial epithelial cells. Fluorescent labeling, CCK-8 and transwell migration assays showed that these EVs were delivered to the trophoblast or parthenogenetic activation embryos to regulate proliferation and migration of trophoblast cells. A total of 305 miRNAs were identified using small RNA sequencing analysis. Functional enrichment analysis showed that miRNAs in these EVs potentially play vital regulatory functions in EV transportation or conceptus implantation. QRT-PCR analysis was used to further verify the RNA-seq data. The findings of this study provide information on the functions of porcine ULF-derived EVs and provide a reference dataset for future translational studies on porcine ULF-derived EVs.


Assuntos
Implantação do Embrião , Vesículas Extracelulares , Animais , Embrião de Mamíferos , Endométrio , Feminino , Camundongos , Gravidez , Suínos , Útero
6.
Ecotoxicol Environ Saf ; 229: 113096, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34952380

RESUMO

Bisphenol A (BPA), an endocrine disruptor, has been replaced by structural analogues including bisphenol S (BPS). BPA and BPS exhibited similar effects regarding reproductive functions. Moreover, metabolic status and lipid metabolism are related to female fertility and could worsen BPS effects. The objective was to determine BPS in vivo effects on folliculogenesis and embryo production after chronic exposure through diet, and the influence of metabolic status in adult ewes. Sixty primiparous 2.5 year-old ewes, undergoing a restricted or well fed diet, were exposed to BPS (0, 4 or 50 µg/kg/day) for at least three months. After hormonal oestrus synchronisation and ovarian stimulation, ewes were subjected to ovum pick-up (OPU) procedures to collect immature oocytes, that underwent in vitro maturation, fertilisation and embryo production. Body weight, body condition score and plasma glucose were higher in well-fed compared to restricted ewes, while plasma NEFA was lower during the 4-5 months after the beginning of the diets. Plasma progesterone levels increased on day 5 before OPU session in well-fed compared to restricted ewes. No effect of BPS dose was observed on follicle population, plasma AMH levels and embryo production numbers and rates. However, a significant diet x BPS dose interaction was reported for cleaved embryos, > 4-cell embryos, blastocyst and early blastocyst numbers, and plasma triiodothyronine levels. Our study showed that a contrasted diet did not affect follicle population nor embryo production in adult ewes but could affect the quality and progesterone secretion of the corpus luteum. Chronic low BPS exposure had no effect on follicular population and oocyte competence. Nevertheless, the significant diet x dose interactions observed on embryo production suggest that BPS effect is modulated by metabolic status. Further studies are required to assess the risk of BPS exposure for public reproductive health.


Assuntos
Oócitos , Sulfonas , Animais , Dieta/veterinária , Embrião de Mamíferos , Feminino , Fenóis , Ovinos
7.
Cell Mol Life Sci ; 79(1): 1, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34910257

RESUMO

Intestinal mesenchymal cells encompass multiple subsets, whose origins, functions, and pathophysiological importance are still not clear. Here, we used the Col6a1Cre mouse, which targets distinct fibroblast subsets and perivascular cells that can be further distinguished by the combination of the CD201, PDGFRα and αSMA markers. Developmental studies revealed that the Col6a1Cre mouse also targets mesenchymal aggregates that are crucial for intestinal morphogenesis and patterning, suggesting an ontogenic relationship between them and homeostatic PDGFRαhi telocytes. Cell depletion experiments in adulthood showed that Col6a1+/CD201+ mesenchymal cells regulate homeostatic enteroendocrine cell differentiation and epithelial proliferation. During acute colitis, they expressed an inflammatory and extracellular matrix remodelling gene signature, but they also retained their properties and topology. Notably, both in homeostasis and tissue regeneration, they were dispensable for normal organ architecture, while CD34+ mesenchymal cells expanded, localised at the top of the crypts, and showed increased expression of villous-associated morphogenetic factors, providing thus evidence for the plasticity potential of intestinal mesenchymal cells. Our results provide a comprehensive analysis of the identities, origin, and functional significance of distinct mesenchymal populations in the intestine.


Assuntos
Colágeno Tipo VI/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Intestinos/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Plasticidade Celular , Proliferação de Células , Colite/induzido quimicamente , Colite/patologia , Colágeno Tipo VI/deficiência , Colágeno Tipo VI/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Intestinos/citologia , Intestinos/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Regeneração
8.
Nat Commun ; 12(1): 7322, 2021 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-34916498

RESUMO

Blastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments. Here, we provide evidence that embryo-like structures can be generated solely based on transcription factor-mediated reprogramming of embryonic stem cells in a simple 3D co-culture system. Embryonic stem cells in these cultures self-organize into elongated, compartmentalized embryo-like structures reflecting aspects of the inner regions of the early post-implantation embryo. Single-cell RNA-sequencing reveals transcriptional profiles resembling epiblast, primitive-/visceral endoderm, and extraembryonic ectoderm of early murine embryos around E4.5-E5.5. In this stem cell-based embryo model, progression from rosette formation to lumenogenesis accompanied by progression from naïve- to primed pluripotency was observed within Epi-like cells. Additionally, lineage specification of primordial germ cells and distal/anterior visceral endoderm-like cells was observed in epiblast- or visceral endoderm-like compartments, respectively. The system presented in this study allows for fast and reproducible generation of embryo-like structures, providing an additional tool to study aspects of early embryogenesis.


Assuntos
Corpos Embrioides/citologia , Desenvolvimento Embrionário , Células-Tronco Embrionárias/citologia , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Reprogramação Celular , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , RNA-Seq
9.
Epigenetics Chromatin ; 14(1): 57, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34930415

RESUMO

BACKGROUND: Dynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently, lactylation has been reported as a novel epigenetic modification that regulates various cellular processes, but its role during early embryogenesis has not been elucidated. RESULTS: We examined nuclear accumulation of H3K23la, H3K18la and pan histone lactylation in mouse oocytes and pre-implantation embryos by immunofluorescence with specific antibodies. All of the three modifications were abundant in GV stage oocytes, and both H3K23la and pan histone lactylation could be detected on the condensed chromosomes of the MII oocytes, while H3K18la were not detected. After fertilization, the nuclear staining of H3K23la, H3K18la and pan histone lactylation was faint in zygotes but homogeneously stained both of the parental pronuclei. The signal remained weak in the early cleavage stage embryos and increased remarkably in the blastocyst stage embryos. Comparison of the embryos cultured in four different conditions with varying concentrations of oxygen found that H3K23la, H3K18la and pan histone lactylation showed similar and comparable staining pattern in embryos cultured in atmospheric oxygen concentration (20% O2), gradient oxygen concentration (5% O2 to 2% O2) and embryos obtained from in vivo, but the modifications were greatly reduced in embryos cultured in hypoxic condition (2% O2). In contrast, nuclear accumulation of H3K18ac or H3K23ac was not significantly affected under hypoxic condition. Moreover, the developmental rate of in vitro cultured embryo was significantly reduced by low oxygen concentration and small molecule inhibition of LDHA activity led to decreased lactate production, as well as reduced histone lactylation and compromised developmental rate. CONCLUSIONS: We provided for the first time the dynamic landscape of H3K23la, H3K18la and pan histone lactylation in oocytes and pre-implantation embryos in mice. Our data suggested that histone lactylation is subjected to oxygen concentration in the culture environment and hypoxic in vitro culture reduces histone lactylation, which in turn compromises developmental potential of pre-implantation embryos in mice.


Assuntos
Desenvolvimento Embrionário , Histonas , Animais , Blastocisto , Embrião de Mamíferos , Feminino , Camundongos , Oócitos , Gravidez , Zigoto
10.
Nat Commun ; 12(1): 7235, 2021 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-34903763

RESUMO

Developmental genes are frequently controlled by multiple enhancers sharing similar specificities. As a result, deletions of such regulatory elements have often failed to reveal their full function. Here, we use the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion of one of its enhancers (Pen). By combining single cell transcriptomics and an in-embryo cell tracing approach, we observe an increased fraction of Pitx1 non/low-expressing cells and a decreased fraction of Pitx1 high-expressing cells. We find that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. This locus mis-activation induces a localized heterochrony and a concurrent loss of irregular connective tissue, eventually leading to a clubfoot phenotype. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes.


Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Box Pareados/genética , Acetilação , Animais , Cromatina/química , Cromatina/metabolismo , Tecido Conjuntivo/crescimento & desenvolvimento , Tecido Conjuntivo/metabolismo , Embrião de Mamíferos , Epigênese Genética , Membro Posterior/citologia , Membro Posterior/embriologia , Membro Posterior/metabolismo , Botões de Extremidades/citologia , Botões de Extremidades/embriologia , Botões de Extremidades/metabolismo , Camundongos , Modelos Genéticos , Fatores de Transcrição Box Pareados/metabolismo , Deleção de Sequência
11.
Nat Commun ; 12(1): 7334, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34921133

RESUMO

The erythroid terminal differentiation program couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. Here we use Epor-/- mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. We find that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. EpoR-regulation of cell size is independent of established red cell size regulation by iron. High erythropoietin (Epo) increases red cell size in wild-type mice and in human volunteers. The increase in mean corpuscular volume (MCV) outlasts the duration of Epo treatment and is not the result of increased reticulocyte number. Our work shows that EpoR signaling alters the relationship between cycling and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress.


Assuntos
Ciclo Celular , Tamanho Celular , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritropoese , Receptores da Eritropoetina/metabolismo , Adulto , Animais , Antígenos CD/metabolismo , Antígenos CD4/metabolismo , Diferenciação Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Embrião de Mamíferos/metabolismo , Eritroblastos/citologia , Eritroblastos/efeitos dos fármacos , Eritroblastos/metabolismo , Eritropoetina/administração & dosagem , Eritropoetina/farmacologia , Feminino , Feto/metabolismo , Voluntários Saudáveis , Humanos , Ferro/metabolismo , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Receptores da Transferrina/metabolismo , Reticulócitos/citologia , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Transdução de Sinais , Proteína bcl-X/metabolismo
12.
PLoS One ; 16(12): e0261357, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34941916

RESUMO

During pregnancy in placental mammals, small numbers of maternal cells (maternal microchimeric cells, or MMc cells) migrate into the fetus and persist decades, or perhaps for the rest of their lives, and higher frequencies of MMc cells are reported to correlate with variety of phenomena, such as immune tolerance, tissue repair, and autoimmune diseases. While detection of these MMc cells is considered in all pregnancies, their frequency differs largely according to tissue type and disease cases, and it remains unclear whether the number of MMc cells differs significantly among embryos in normal pregnancies. Here, for the first time, we developed a whole embryonic detection method for MMc cells using transgenic mice and counted live MMc cells in each individual embryo. Using this technique, we found that the number of MMc cells was comparable in most of the analyzed embryos; however, around 500 times higher number of MMc cells was detected in one embryo at the latest stage. This result suggests that the number of MMc cells could largely differ in rare cases with unknown underlying mechanisms. Our methodology provides a basis for testing differences in the numbers of MMc cells among individual embryos and for analyzing differences in MMc cell type repertoires in future studies. These data could provide a hint toward understanding the mechanisms underlying the variety of apparently inconsistent MMc-related phenomena.


Assuntos
Quimerismo/embriologia , Animais , Quimerismo/estatística & dados numéricos , Embrião de Mamíferos/imunologia , Embrião de Mamíferos/metabolismo , Eutérios/metabolismo , Feminino , Feto , Tolerância Imunológica/imunologia , Troca Materno-Fetal/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Placenta , Gravidez
13.
J Biomed Opt ; 26(11)2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34773396

RESUMO

SIGNIFICANCE: Real-time monitoring of the heart rate and blood flow is crucial for studying cardiovascular dysfunction, which leads to cardiovascular diseases. AIM: This study aims at in-depth understanding of high-speed cardiovascular dynamics in a zebrafish embryo model for various biomedical applications via frequency-comb-referenced quantitative phase imaging (FCR-QPI). APPROACH: Quantitative phase imaging (QPI) has emerged as a powerful technique in the field of biomedicine but has not been actively applied to the monitoring of circulatory/cardiovascular parameters, due to dynamic speckles and low frame rates. We demonstrate FCR-QPI to measure heart rate and blood flow in a zebrafish embryo. FCR-QPI utilizes a high-speed photodetector instead of a conventional camera, so it enables real-time monitoring of individual red blood cell (RBC) flow. RESULTS: The average velocity of zebrafish's RBCs was measured from 192.5 to 608.8 µm / s at 24 to 28 hour-post-fertilization (hpf). In addition, the number of RBCs in a pulsatile blood flow was revealed to 16 cells/pulse at 48 hpf. The heart rates corresponded to 94 and 142 beats-per-minute at 24 and 48 hpf. CONCLUSIONS: This approach will newly enable in-depth understanding of the cardiovascular dynamics in the zebrafish model and possible usage for drug discovery applications in biomedicine.


Assuntos
Hemodinâmica , Peixe-Zebra , Animais , Diagnóstico por Imagem , Embrião de Mamíferos , Embrião não Mamífero , Frequência Cardíaca
14.
Nat Commun ; 12(1): 5855, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615869

RESUMO

Karyotype alterations have emerged as on-target complications from CRISPR-Cas9 genome editing. However, the events that lead to these karyotypic changes in embryos after Cas9-treatment remain unknown. Here, using imaging and single-cell genome sequencing of 8-cell stage embryos, we track both spontaneous and Cas9-induced karyotype aberrations through the first three divisions of embryonic development. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development.


Assuntos
Sistemas CRISPR-Cas , Estruturas Cromossômicas , Edição de Genes/métodos , Instabilidade Genômica , Animais , Segregação de Cromossomos , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Cariótipo , Camundongos , Sequenciamento Completo do Genoma
15.
Theriogenology ; 176: 149-162, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34619436

RESUMO

Embryonic implantation is a complex reproductive physiological process in mammals. Although several endometrial proteins affecting embryonic implantation have been reported in the past, there are still potential endometrial proteins that have been neglected, and their specific regulatory mechanisms are unclear. This study demonstrated that protein phosphatase 2A regulatory subunit B55α (PPP2R2A) served as a novel regulator in medication of sheep embryonic implantation in vitro. Our results showed that sheep PPP2R2A encoded 447 amino acids and shared 91.74%-92.36% amino acid sequences with its orthologs compared with other species. Meanwhile, PPP2R2A was widely expressed in sheep uterine tissues, and it could regulate the expression levels of key regulators of embryonic implantation in endometrial stromal cells (ESCs). Knockdown of PPP2R2A significantly inhibited cell proliferation by blocking cell cycle transfer G0/G1 into S phase accompanied by downregulation of CDK2, CDK4, CCND1, CCNE1 and upregulation of P21. In contrast to PPP2R2A overexpression, PPP2R2A interference greatly promoted cell apoptosis and the expression of BAX, CASP3, CASP9 and BAX/BCL-2. Taken together, these results suggest that PPP2R2A, as a novel regulatory factor, affects embryonic implantation via regulating the proliferation and apoptosis of Hu sheep ESCs in vitro.


Assuntos
Apoptose , Proteína Fosfatase 2 , Ovinos , Animais , Proliferação de Células , Implantação do Embrião , Embrião de Mamíferos , Células Estromais
16.
Theriogenology ; 176: 183-187, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34624812

RESUMO

The aim of the study was to compare three methods of reducing twin pregnancy in mares to maintain a single pregnancy. As multiple pregnancies in mare are always undesirable, early ultrasound diagnosis makes possible management of twin pregnancies and extra embryo removal. In years 2010-2018, 16494 mares were sonographically tested for early pregnancy, finding 868 cases of twins (471 bilateral and 397 unilateral). 260 mares with a confirmed bilateral tween pregnancy were subjected to manual crushing of one embryo and administration of flunixin at a dose of 1.1 mg/kg BW. 186 mares were subjected only to the embryo crushing procedure. 25 mares from this group were on a restrictive diet. In the unilateral twin pregnancy mare group, 62 were subjected to manual embryo reduction with simultaneous treatment with flunixin, 60 had only manual embryonic vesicle crush and 210 had a restrictive diet. Determination of success, measured as the development of a single pregnancy, were monitored 2 weeks after the procedure, between the 50th and 60th day of pregnancy and after the 90th day of pregnancy. In general, warm-blooded mares were more prone to a twin pregnancy, and at the same time, all the procedures used to reduce it to a single pregnancy caused a greater risk of losing both embryos than in the case of cold-blooded mares. The beneficial effect of administering flunixin after manual removal of one embryo on the maintenance of the other has been experimentally proven in both unilateral and bilateral twin pregnancy.


Assuntos
Redução de Gravidez Multifetal , Prenhez , Animais , Embrião de Mamíferos , Feminino , Cavalos , Gravidez , Redução de Gravidez Multifetal/veterinária , Gravidez Múltipla , Estudos Retrospectivos , Ultrassonografia
17.
Nat Commun ; 12(1): 5736, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593792

RESUMO

Despite the emerging importance of reactive electrophilic drugs, deconvolution of their principal targets remains difficult. The lack of genetic tractability/interventions and reliance on secondary validation using other non-specific compounds frequently complicate the earmarking of individual binders as functionally- or phenotypically-sufficient pathway regulators. Using a redox-targeting approach to interrogate how on-target binding of pleiotropic electrophiles translates to a phenotypic output in vivo, we here systematically track the molecular components attributable to innate immune cell toxicity of the electrophilic-drug dimethyl fumarate (Tecfidera®). In a process largely independent of canonical Keap1/Nrf2-signaling, Keap1-specific modification triggers mitochondrial-targeted neutrophil/macrophage apoptosis. On-target Keap1-ligand-engagement is accompanied by dissociation of Wdr1 from Keap1 and subsequent coordination with cofilin, intercepting Bax. This phagocytic-specific cell-killing program is recapitulated by whole-animal administration of dimethyl fumarate, where individual depletions of the players identified above robustly suppress apoptosis.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Fumarato de Dimetilo/farmacologia , Imunossupressores/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Embrião de Mamíferos , Embrião não Mamífero , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imunidade Inata/efeitos dos fármacos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Peixe-Zebra
18.
Nat Commun ; 12(1): 5771, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599190

RESUMO

Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.


Assuntos
Elementos Facilitadores Genéticos , Células Germinativas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animais , Diferenciação Celular , Cromatina/metabolismo , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica , Células Germinativas/citologia , Camadas Germinativas/citologia , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Mutação/genética , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , RNA-Seq , Análise de Célula Única , Sítio de Iniciação de Transcrição , Transcrição Genética
19.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639209

RESUMO

The process of freezing cells or tissues and depositing them in liquid nitrogen at -196 °C is called cryopreservation. Sub-zero temperature is not a physiological condition for cells and water ice crystals represent the main problem since they induce cell death, principally in large cells like oocytes, which have a meiotic spindle that degenerates during this process. Significantly, cryopreservation represents an option for fertility preservation in patients who develop gonadal failure for any condition and those who want to freeze their germ cells for later use. The possibility of freezing sperm, oocytes, and embryos has been available for a long time, and in 1983 the first birth with thawed oocytes was achieved. From the mid-2000s forward, the use of egg vitrification through intracytoplasmic sperm injection has improved pregnancy rates. Births using assisted reproductive technologies (ART) have some adverse conditions and events. These risks could be associated with ART procedures or related to infertility. Cryopreservation generates changes in the epigenome of gametes and embryos, given that ART occurs when the epigenome is most vulnerable. Furthermore, cryoprotective agents induce alterations in the integrity of germ cells and embryos. Notably, cryopreservation extensively affects cell viability, generates proteomic profile changes, compromises crucial cellular functions, and alters sperm motility. This technique has been widely employed since the 1980s and there is a lack of knowledge about molecular changes. The emerging view is that molecular changes are associated with cryopreservation, affecting metabolism, cytoarchitecture, calcium homeostasis, epigenetic state, and cell survival, which compromise the fertilization in ART.


Assuntos
Cálcio/metabolismo , Criopreservação/normas , Embrião de Mamíferos/citologia , Epigênese Genética , Células Germinativas/citologia , Infertilidade/terapia , Proteoma/metabolismo , Sobrevivência Celular , Crioprotetores/química , Feminino , Preservação da Fertilidade/normas , Fertilização In Vitro , Células Germinativas/metabolismo , Humanos , Infertilidade/metabolismo , Infertilidade/patologia , Masculino , Oócitos/citologia , Oócitos/metabolismo , Gravidez , Espermatozoides/citologia , Espermatozoides/metabolismo
20.
Int J Mol Sci ; 22(19)2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34638777

RESUMO

The mechanisms of neural crest cell induction and specification are highly conserved among vertebrate model organisms, but how similar these mechanisms are in mammalian neural crest cell formation remains open to question. The zinc finger of the cerebellum 1 (ZIC1) transcription factor is considered a core component of the vertebrate gene regulatory network that specifies neural crest fate at the neural plate border. In mouse embryos, however, Zic1 mutation does not cause neural crest defects. Instead, we and others have shown that murine Zic2 and Zic5 mutate to give a neural crest phenotype. Here, we extend this knowledge by demonstrating that murine Zic3 is also required for, and co-operates with, Zic2 and Zic5 during mammalian neural crest specification. At the murine neural plate border (a region of high canonical WNT activity) ZIC2, ZIC3, and ZIC5 function as transcription factors to jointly activate the Foxd3 specifier gene. This function is promoted by SUMOylation of the ZIC proteins at a conserved lysine immediately N-terminal of the ZIC zinc finger domain. In contrast, in the lateral regions of the neurectoderm (a region of low canonical WNT activity) basal ZIC proteins act as co-repressors of WNT/TCF-mediated transcription. Our work provides a mechanism by which mammalian neural crest specification is restricted to the neural plate border. Furthermore, given that WNT signaling and SUMOylation are also features of non-mammalian neural crest specification, it suggests that mammalian neural crest induction shares broad conservation, but altered molecular detail, with chicken, zebrafish, and Xenopus neural crest induction.


Assuntos
Embrião de Mamíferos/embriologia , Crista Neural/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Transcrição Genética , Via de Sinalização Wnt , Animais , Embrião de Mamíferos/citologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Camundongos Transgênicos , Crista Neural/citologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética
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