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1.
Biol Reprod ; 107(6): 1439-1451, 2022 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-36130203

RESUMO

Egg activation in mammals is triggered by oscillations in egg intracellular calcium (Ca2+) level. Ca2+ oscillation patterns can be modified in vitro by changing the ionic composition of culture media or in vivo by conditions affecting mitochondrial function, such as obesity and inflammation. In mice, disruption of Ca2+ oscillations in vitro impacts embryo development and offspring growth. Here we tested the hypothesis that, even without in vitro manipulation, abnormal Ca2+ signaling following fertilization impacts offspring growth. Plasma membrane Ca2+ ATPases (PMCA) extrude cytosolic Ca2+ to restore Ca2+ homeostasis. To disrupt Ca2+ signaling in vivo, we conditionally deleted PMCA1 (cKO) in oocytes. As anticipated, in vitro fertilized cKO eggs had increased Ca2+ exposure relative to controls. To assess the impact on offspring growth, cKO females were mated to wild type males to generate pups that had high Ca2+ exposure at fertilization. Because these offspring would be heterozygous, we also tested the impact of global PMCA1 heterozygosity on offspring growth. Control heterozygous pups that had normal Ca2+ at fertilization were generated by mating wild type females to heterozygous males; these control offspring weighed significantly less than their wild type siblings. However, heterozygous offspring from cKO eggs (and high Ca2+ exposure) were larger than heterozygous controls at 12 week-of-age and males had altered body composition. Our results show that global PMCA1 haploinsufficiency impacts growth and support that abnormal Ca2+ signaling after fertilization in vivo has a long-term impact on offspring weight. These findings are relevant for environmental and medical conditions affecting Ca2+ handling and for design of culture conditions and procedures for domestic animal and human assisted reproduction.


Assuntos
Sinalização do Cálcio , Cálcio , Masculino , Feminino , Camundongos , Humanos , Animais , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Fertilização/fisiologia , Zigoto/metabolismo , Oócitos/metabolismo , Mamíferos/metabolismo
2.
Reproduction ; 159(6): 767-778, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32240977

RESUMO

CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development.


Assuntos
DNA Helicases/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Proteínas Nucleares/metabolismo , Fator de Transcrição AP-2/metabolismo , Fatores de Transcrição/metabolismo , Animais , Bovinos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Helicases/genética , Fertilização in vitro/veterinária , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/veterinária , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Fator de Transcrição AP-2/genética , Fatores de Transcrição/genética
3.
Nat Genet ; 56(4): 697-709, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38509386

RESUMO

In mice, exit from the totipotent two-cell (2C) stage embryo requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here we demonstrate that the 2C-specific transcription factor double homeobox protein (DUX) mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing. We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression. Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the TRIM superfamily involved in gene silencing, and colocalizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in mouse embryonic stem cells increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts leading to early developmental arrest. Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates.


Assuntos
Genes Homeobox , Proteínas de Homeodomínio , Células-Tronco Embrionárias Murinas , Fatores de Transcrição , Animais , Camundongos , Diferenciação Celular , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo
4.
Cell Rep ; 42(10): 113232, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37824328

RESUMO

TRPM7 (transient receptor potential cation channel subfamily M member 7) is a chanzyme with channel and kinase domains essential for embryo development. Using gamete-specific Trpm7-null lines, we report that TRPM7-mediated Mg2+ influx is indispensable for reaching the blastocyst stage. TRPM7 is expressed dynamically from gametes to blastocysts; displays stage-specific localization on the plasma membrane, cytoplasm, and nucleus; and undergoes cleavage that produces C-terminal kinase fragments. TRPM7 underpins Mg2+ homeostasis, and excess Mg2+ but not Zn2+ or Ca2+ overcomes the arrest of Trpm7-null embryos; expressing Trpm7 mRNA restores development, but mutant versions fail or are partially rescued. Transcriptomic analyses of Trpm7-null embryos reveal an abundance of oxidative stress-pathway genes, confirmed by mitochondrial dysfunction, and a reduction in transcription factor networks essential for proliferation; Mg2+ supplementation corrects these defects. Hence, TRPM7 underpins Mg2+ homeostasis in preimplantation embryos, prevents oxidative stress, and promotes gene expression patterns necessary for developmental progression and cell-lineage specification.


Assuntos
Desenvolvimento Embrionário , Magnésio , Canais de Cátion TRPM , Animais , Camundongos , Citoplasma/metabolismo , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Canais de Cátion TRPM/metabolismo , Magnésio/metabolismo
5.
Methods Mol Biol ; 2495: 129-148, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35696032

RESUMO

The CRISPR-on system is a programmable, simple, and versatile gene activator that has proven to be efficient in cultured cells from several species and in bovine embryos. This technology allows for the precise and specific activation of single endogenous gene expression and also multiplexed gene expression in a simple fashion. Therefore, CRISPR-on has unique advantages over other activator systems and a wide adaptability for studies in basic and applied science, such as cell reprogramming and cell fate differentiation for regenerative medicine.In this chapter, we describe the materials and methods of the CRISPR-on system for activation of the endogenous SMARCA4 expression in bovine embryos.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Animais , Bovinos , Diferenciação Celular , Linhagem Celular , Reprogramação Celular/genética
6.
Front Cell Dev Biol ; 9: 762057, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34805168

RESUMO

Superovulation is a common approach to maximize the number of eggs available for either clinical assisted reproductive technologies or experimental animal studies. This procedure provides supraphysiological amounts of gonadotropins to promote continued growth and maturation of ovarian follicles that otherwise would undergo atresia. There is evidence in mice, cows, sheep, and humans that superovulation has a detrimental impact on the quality of the resulting ovulated eggs or embryos. Here we tested the hypothesis that eggs derived from superovulation have a reduced capacity to support calcium oscillations, which are a critical factor in the success of embryo development. Eggs were obtained from mice that were either naturally cycling or underwent a standard superovulation protocol. The eggs were either parthenogenetically activated using strontium or fertilized in vitro while undergoing monitoring of calcium oscillatory patterns. Following parthenogenetic activation, superovulated eggs had a slightly delayed onset and longer duration of the first calcium transient, but no differences in oscillation persistence, frequency, or total calcium signal. However, in vitro fertilized superovulated eggs had no differences in any of these measures of calcium oscillatory behavior relative to spontaneously ovulated eggs. These findings indicate that although subtle differences in calcium signaling can be detected following parthenogenetic activation, superovulation does not disrupt physiological calcium signaling at fertilization, supporting the use of this method for both clinical and experimental purposes.

7.
Cell Reprogram ; 23(5): 277-289, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34648384

RESUMO

Somatic cell nuclear transfer (SCNT) is a method with unique ability to reprogram the epigenome of a fully differentiated cell. However, its efficiency remains extremely low. In this work, we assessed and combined two simple strategies to improve the SCNT efficiency in the bovine. These are the use of less-differentiated donor cells to facilitate nuclear reprogramming and the embryo aggregation (EA) strategy that is thought to compensate for aberrant epigenome reprogramming. We carefully assessed the optimal time of EA by using in vitro-fertilized (IVF) embryos and evaluated whether the use of adipose-derived mesenchymal stem cells (ASCs) as donor for SCNT together with EA improves the blastocyst rates and quality. Based on our results, we determined that the EA improves the preimplantation embryo development per well of IVF and SCNT embryos. We also demonstrated that day 0 (D0) is the optimal aggregation time that leads to a single blastocyst with uniform distribution of the original blastomeres. This was confirmed in bovine IVF embryos and then, the optimal condition was translated to SCNT embryos. Notably, the relative expression of the trophectoderm (TE) marker KRT18 was significantly different between aggregated and nonaggregated ASC-derived embryos. In the bovine, no effect of the donor cell is observed on the developmental rate, or the embryo quality. Therefore, no synergistic effect of the use of both strategies is observed. Our results suggest that EA at D0 is a simple and accessible strategy that improves the blastocyst rate per well in bovine SCNT and IVF embryos and influence the expression of a TE-related marker. The aggregation of two ASC-derived embryos seems to positively affect the embryo quality, which may improve the postimplantation development.


Assuntos
Blastocisto/citologia , Clonagem de Organismos/veterinária , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Células-Tronco Mesenquimais/citologia , Animais , Bovinos , Embrião de Mamíferos/química , Feminino , Fertilização in vitro , Gravidez
8.
Open Biol ; 10(7): 200118, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673518

RESUMO

Calcium (Ca2+) signals initiate egg activation across the animal kingdom and in at least some plants. These signals are crucial for the success of development and, in the case of mammals, health of the offspring. The mechanisms associated with fertilization that trigger these signals and the molecules that regulate their characteristic patterns vary widely. With few exceptions, a major contributor to fertilization-induced elevation in cytoplasmic Ca2+ is release from endoplasmic reticulum stores through the IP3 receptor. In some cases, Ca2+ influx from the extracellular space and/or release from alternative intracellular stores contribute to the rise in cytoplasmic Ca2+. Following the Ca2+ rise, the reuptake of Ca2+ into intracellular stores or efflux of Ca2+ out of the egg drive the return of cytoplasmic Ca2+ back to baseline levels. The molecular mediators of these Ca2+ fluxes in different organisms include Ca2+ release channels, uptake channels, exchangers and pumps. The functions of these mediators are regulated by their particular activating mechanisms but also by alterations in their expression and spatial organization. We discuss here the molecular basis for modulation of Ca2+ signalling at fertilization, highlighting differences across several animal phyla, and we mention key areas where questions remain.


Assuntos
Sinalização do Cálcio/genética , Cálcio/metabolismo , Fertilização/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Retículo Endoplasmático/genética , Humanos
9.
Dev Cell ; 53(5): 545-560.e7, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32442396

RESUMO

Embryonic genome activation (EGA) is orchestrated by an intrinsic developmental program initiated during oocyte maturation with translation of stored maternal mRNAs. Here, we show that tankyrase, a poly(ADP-ribosyl) polymerase that regulates ß-catenin levels, undergoes programmed translation during oocyte maturation and serves an essential role in mouse EGA. Newly translated TNKS triggers proteasomal degradation of axin, reducing targeted destruction of ß-catenin and promoting ß-catenin-mediated transcription of target genes, including Myc. MYC mediates ribosomal RNA transcription in 2-cell embryos, supporting global protein synthesis. Suppression of tankyrase activity using knockdown or chemical inhibition causes loss of nuclear ß-catenin and global reductions in transcription and histone H3 acetylation. Chromatin and transcriptional profiling indicate that development arrests prior to the mid-2-cell stage, mediated in part by reductions in ß-catenin and MYC. These findings indicate that post-transcriptional regulation of tankyrase serves as a ligand-independent developmental mechanism for post-translational ß-catenin activation and is required to complete EGA.


Assuntos
Blastocisto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Tanquirases/metabolismo , beta Catenina/genética , Animais , Blastocisto/citologia , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Tanquirases/genética , Regulação para Cima , beta Catenina/metabolismo
10.
Plant Signal Behav ; 13(2): e1432956, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29452030

RESUMO

BACKGROUND: Small monomeric GTPases act as molecular switches in several processes that involve polar cell growth, participating mainly in vesicle trafficking and cytoskeleton rearrangements. This gene superfamily has largely expanded in plants through evolution as compared with other Kingdoms, leading to the suggestion that members of each subfamily might have acquired new functions associated to plant-specific processes. Legume plants engage in a nitrogen-fixing symbiotic interaction with rhizobia in a process that involves polar growth processes associated with the infection throughout the root hair. To get insight into the evolution of small GTPases associated with this process, we use a comparative genomic approach to establish differences in the Ras GTPase superfamily between legume and non-legume plants. RESULTS: Phylogenetic analyses did not show clear differences in the organization of the different subfamilies of small GTPases between plants that engage or not in nodule symbiosis. Protein alignments revealed a strong conservation at the sequence level of small GTPases previously linked to nodulation by functional genetics. Interestingly, one Rab and three Rop proteins showed conserved amino acid substitutions in legumes, but these changes do not alter the predicted conformational structure of these proteins. Although the steady-state levels of most small GTPases do not change in response to rhizobia, we identified a subset of Rab, Rop and Arf genes whose transcript levels are modulated during the symbiotic interaction, including their spatial distribution along the indeterminate nodule. CONCLUSIONS: This study provides a comprehensive study of the small GTPase superfamily in several plant species. The genetic program associated to root nodule symbiosis includes small GTPases to fulfill specific functions during infection and formation of the symbiosomes. These GTPases seems to have been recruited from members that were already present in common ancestors with plants as distant as monocots since we failed to detect asymmetric evolution in any of the subfamily trees. Expression analyses identified a number of legume members that can have undergone neo- or sub-functionalization associated to the spatio-temporal transcriptional control during the onset of the symbiotic interaction.


Assuntos
Fabaceae/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fabaceae/genética , Genômica , Proteínas Monoméricas de Ligação ao GTP/classificação , Proteínas Monoméricas de Ligação ao GTP/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Simbiose/genética , Simbiose/fisiologia
11.
Theriogenology ; 85(7): 1297-311.e2, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26838464

RESUMO

Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the piggyBac (PB) and sleeping beauty (SB) transposon systems were assessed for stable gene transfer into the cattle genome. Bovine fibroblasts were transfected either with a helper-independent PB system or a binary SB system. Both transposons were highly active in bovine cells increasing the efficiency of DNA integration up to 88 times over basal nonfacilitated integrations in a colony formation assay. SB transposase catalyzed multiplex transgene integrations in fibroblast cells transfected with the helper vector and two donor vectors carrying different transgenes (fluorophore and neomycin resistance). Stably transfected fibroblasts were used for SCNT and on in vitro embryo culture, morphologically normal blastocysts that expressed the fluorophore were obtained with both transposon systems. The data indicate that transposition is a feasible approach for genetic engineering in the cattle genome.


Assuntos
Bovinos/genética , Elementos de DNA Transponíveis/genética , Vetores Genéticos/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , Fibroblastos , Técnicas de Transferência Nuclear , Transfecção , Transposases
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