The expression of a subset of transcription factors is enriched in early preimplantation embryos, which contributes to their cellular plasticity. RONIN, NANOG and its associated proteins are PluripotencyAssociated Transcription Factors (PATF) that control relevant downstream pathways in pluripotent stem cells, but their activity in early embryos remained less understood. The work was aimed to determine the expression of RONIN and four NANOG-associated PATFs in goat preimplantation embryos. Goat embryos were produced in vitro by parthenogenetic activation. Gene transcripts of cleavage-stage embryos were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR), while blastocysts were analyzed by both RT-PCR and quantitative RT-PCR (RT-qPCR) assays. Gene transcripts of ZFP281, NAC1, and NR0B1 were detected in cleavage-stage embryos, while RONIN and OCT4 were not found expressed. Detection in blastocysts by RT-PCR confirmed the activity of NR0B1, RONIN, and OCT4. Moreover, all five PATF were detected in blastocysts by RT-qPCR (ZFP281, NAC1, RONIN, OCT4, and NR0B1). In conclusion, RONIN and NANOG-associated proteins are active during goat parthenogenetic preimplantation development and hold stage-specific expression patterns.Keywords: Capra hircus; caprine; embryogenesis; RT-PCR; RT-qPCR; mRNA. ResumoA expressão de um conjunto de fatores de transcrição é aumentada em embriões antes da implantação, fato que contribui para a sua plasticidade celular. RONIN, NANOG e suas proteínas associadas são Fatores de transcrição relacionados à pluripotência (FTRP) que controlam vias de sinalização importantes para células-tronco pluripotentes, embora suas atividades em embriões permaneçam menos compreendidas. O objetivo do trabalho foi determinar a expressão de RONIN e quatro FTRPs associados ao NANOG em embriões caprinos. Os embriões foram produzidos in vitro por ativação partenogenética. Embriões na fase de clivagem foram investigados por transcrição reversa, seguida da reação da reação em cadeia da polimerase (RT-PCR), enquanto que os blastocistos foram analisados por RT-PCR e por RT-PCR quantitativa (RT-qPCR). Os transcritos de ZFP281, NAC1 e NR0B1 foram detectados em embriões clivados, enquanto que RONIN e OCT4 não foram encontrados. A detecção em blastocistos pela RT-PCR confirmou a atividade de NR0B1, RONIN e OCT4. Além disso, os cinco FTRPS foram detectados em blastocistos pela RT-qPCR (ZFP281, NAC1, RONIN, OCT4 e NR0B1). Em conclusão, RONIN e os FTRP associados ao NANOG são ativos durante o desenvolvimento pré-implantacional de caprinos e apresentam padrão de expressão estádio-específica.
Summary Housekeeping genes (HKG) are paramount for accurate gene expression analysis during preimplantation development. Markedly, quantitative reverse transcription polymerase chain reaction (RT-qPCR) in ovine embryos currently lacks HKGs. Therefore, we tested 11 HKGs for RT-qPCR normalization during ovine parthenogenetic preimplantation development. Seven HKGs reached the qPCR efficiency threshold (97.20–105.96%), with correlation coefficients ranging from −0.922 to −0.998 and slopes from −3.22 to −3.59. GeNorm ranked glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-binding protein (TBP) as the best HKG pair, while H3 histone, family 3A (H3F3A) was the third HKG. Relative gene expression was measured for zinc finger protein X-linked (ZFX) and developmental pluripotency-associated 3 (DPPA3) transcripts during ovine parthenogenetic preimplantation development. ZFX did not show any transcript abundance fluctuation among oocytes, cleavage-stage embryos, and morulae. DPPA3 transcript abundance was also similar among all developmental stages, therefore suggesting that it may not display a maternal gene expression profile. In silico analysis of ovine DPPA3 mRNA and protein showed high conservation to bovine orthologues. However, DPPA3 orthologues differed in regulatory motifs. In conclusion, GAPDH, TBP and H3F3A are stable HKGs in ovine parthenogenetic embryos and allow accurate RT-qPCR-based gene expression analysis.
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