SummaryPluripotency-associated transcription factors (PATFs) modulate gene expression during early mammalian embryogenesis. Despite a strong understanding of PATFs during mouse embryogenesis, limited progress has been made in ruminants. This work aimed to describe the temporal expression of eight PATFs during both sheep and cattle preimplantation development. Transcript availability of PATFs was evaluated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in eggs, cleavage-stage embryos, morulae, and blastocysts. Transcripts of five genes were detected in all developmental stages of both species (KLF5, OCT4, RONIN, ZFP281, and ZFX). Furthermore, CMYC was detected in all cattle samples but was found from cleavage-stage onwards in sheep. In contrast, NR0B1 was detected in all sheep samples but was not detected in cattle morulae. GLIS1 displayed the most significant variation in temporal expression between species, as this PATF was only detected in cattle eggs and sheep cleavage-stage embryos and blastocysts. In silico analysis suggested that cattle and sheep PATFs share similar size, isometric point and molecular weight. A phenetic analysis showed two patterns of PATF clustering between cattle and sheep, among several mammalian species. In conclusion, the temporal expression of pluripotency-associated transcription factors differs between sheep and cattle, suggesting species-specific regulation during preimplantation development.
The extent to which mammalian cells share similar transcriptomes remains unclear. Notwithstanding, such cross-species gene expression inquiries have been scarce for defined cell types and most lack the dissection of gene regulatory landscapes. Therefore, the work was aimed to determine C-MYC relative expression across mammalian fibroblasts (Ovis aries and Bos taurus) via cross-species RT-qPCR and comprehensively explore its regulatory landscape by in silico tools. The prediction of transcription factor binding sites in C-MYC and its 2.5 kb upstream sequence revealed substantial variation, thus indicating evolutionary-driven re-wiring of cis-regulatory elements. C-MYC and its downstream target TBX3 were up-regulated in Bos taurus fibroblasts. The relative expression of C-MYC regulators [RONIN (also known as THAP11), RXRβ, and TCF3] and the C-MYC-associated transcript elongation factor CDK9 did not differ between species. Additional in silico analyses suggested Bos taurus-specific C-MYC exonization, alternative splicing, and binding sites for non-coding RNAs. C-MYC protein orthologs were highly conserved, while variation was in the transactivation domain and the leucine zipper motif. Altogether, mammalian fibroblasts display evolutionary-driven C-MYC relative expression that should be instructive for understanding cellular physiology, cellular reprogramming, and C-MYC-related diseases.
Embriões antes da implantação possuem células pluripotentes, ou seja, células que apresentam a capacidade de se diferenciar em todos os tecidos que compõem o feto. O controle da pluripotência em nível molecular é estabelecido por diversos fatores, entre eles, os genes relacionados à pluripotência (GRPs). Estes genes contribuem para inibição do processo de diferenciação celular e manutenção da viabilidade de células pluripotentes. No entanto, apesar do crescente conhecimento sobre as funções dos GRPs em camundongos e humanos, pouco se conhece sobre a expressão espaço-temporal e funções dos GRPs em outras espécies, incluindo ovinos. Evidências em bovinos, humanos e camundongos demonstram que GRPs podem apresentar mecanismos de ação diferentes entre as espécies. O objetivo da revisão foi analisar a atividade dos GRPs em ovinos através do perfil de expressão espaço-temporal e funções, bem como apresentar alternativas para acelerar o entendimento da pluripotência na espécie.
Quantitative reverse transcription PCR (RT-qPCR) remains as an accurate approach for gene expression analysis but requires labor-intensive validation of reference genes using species-specific primers. To ease such demand, the aim was to design and test a multi-species primer set to validate reference genes for inter-genus RT-qPCR gene expression analysis. Primers were designed for ten housekeeping genes using transcript sequences of various livestock species. All ten gene transcripts were detected by RT-PCR in
Bos taurus
(cattle),
Bubalus bubalis
(buffaloes),
Capra hircus
(goats), and
Ovis aries
(sheep) cDNA. Primer efficiency was attained for eight reference genes using
B
.
taurus—O
.
aries
fibroblast cDNA (95.54–98.39%). The RT-qPCR data normalization was carried out for
B
.
taurus
vs.
O
.
aries
relative gene expression using Bestkeeper, GeNorm, Norm-finder, Delta CT method, and RefFinder algorithms. Validation of inter-genus RT-qPCR showed up-regulation of
TLR4
and
ZFX
gene transcripts in
B
.
taurus
fibroblasts, irrespectively of normalization conditions (two, three, or four reference genes).
In silico
search in mammalian transcriptomes showed that the multi-species primer set is expected to amplify transcripts of at least two distinct loci in 114 species, and 79 species would be covered by six or more primers. Hence, a multi-species primer set allows for inter-genus gene expression analysis between
O
.
aries
and
B
.
taurus
fibroblasts and further reveals species-specific gene transcript abundance of key transcription factors.
Evaluación de los días de recolección y transferencia de embriones sobre las tasas de preñez en yeguas Mangalarga Marchador durante la estación de monta Avaliação do dia da colheita e da transferência de embriões equino da raça Mangalarga Marchador durante a estação de monta
Pluripotency-associated transcription factors (PATF) play significant roles during early embryogenesis and in embryonic stem (ES) cells, such as control of cell-cycle progression, modulation of cellular metabolism, and transcriptional control of differentiation-inducing factors. The review aims to describe the current understanding of how these PATFs contribute to the early embryo and the ES-cell phenotypes. By a selection of representative examples of such PATFs, their roles are described, and some interesting questions are presented concerning their activity in pluripotent cells which have yet to be addressed.Keywords: embryology; pluripotent; preimplantation development; totipotency.
ResumoFatores de transcrição relacionados à pluripotência (FTRP) apresentam importantes funções durante a embriogênese inicial e em células-tronco embrionárias (CTE), como o controle da progressão do ciclo celular, modulação do metabolismo celular e controle transcricional de fatores indutores de diferenciação celular. O objetivo da revisão foi descrever o conhecimento vigente sobre como estes FTRPs contribuem para o fenótipo dos embriões e CTEs. Através da seleção de exemplos representativos destes FTRPs, suas funções são descritas e algumas perguntas interessantes são apresentadas considerando suas atividades em células pluripotentes, mas que ainda não foram devidamente respondidas.Palavras-Chave: embriologia; pluripotente; desenvolvimento pré-implantacional; totipotência.
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