Activity of non-canonical pluripotency-associated transcription factors in goat cumulus-oocyte complexes

Moura, Marcelo Tigre; Silva, Roberta L.O.; Cantanhêde, Ludymila Furtado; Silva, Jéssica B.; Ferreira-Silva, José Carlos; Silva, Priscila G. C.; Ramos-Deus, Pamela; Pandolfi, Valesca; Kido, Ederson Akio; Benko‐Iseppon, Ana Maria; Oliveira, Marcos Antônio Lemos

doi:10.1016/j.livsci.2018.03.018

Cited by 7 publications

(7 citation statements)

References 51 publications

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“…aries inter-genus RT-qPCR as assessed by five algorithms. Fourthly, some primers have been used for intra-specific RT-PCR in eggs and early embryos of livestock species [42,43].…”

Section: Discussionmentioning

confidence: 99%

Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set

et al. 2019

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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.

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“…aries inter-genus RT-qPCR as assessed by five algorithms. Fourthly, some primers have been used for intra-specific RT-PCR in eggs and early embryos of livestock species [42,43].…”

Section: Discussionmentioning

confidence: 99%

Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set

et al. 2019

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“…The cumulus-oocyte complexes (COCs) were retrieved from 2 to 8 mm follicles in TCM-199 medium with Hank's salts (Sigma Aldrich) supplemented with 2.0 mM L-glutamine (Sigma Aldrich), 10% fetal bovine serum (FBS; Gibco) and 0.05 mg mL À1 gentamicin sulphate (Gibco). COCs were selected based on at least three layers of CCs and an evenly granulated oocyte cytoplasm (Moura et al, 2018). IVM medium was formulated with TCM-199 with Earle's salts, 2.0 mM L-glutamine, 10% FBS, 10 IU mL À1 penicillin (Gibco), 10 mg mL À1 streptomycin (Gibco), and 0.05 IU mL À1 porcine FSH (FSHþ; Sigma Aldrich) or vehicle control (CTL).…”

Section: Ovary Collection and In Vitro Maturation (Ivm)mentioning

confidence: 99%

MYC integrates FSH signalling networks in cumulus cells during bovine oocyte maturation

Cantanhêde

Moura

Silva

et al. 2022

AVet

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Follicle-stimulating hormone (FSH) contributes to the acquisition of oocyte competence by modulating signalling pathways in cumulus cells (CCs), albeit much less is known about transcription factors (TFs) that orchestrate the downstream transcriptional changes. This work allowed to prospect TFs involved in FSH-mediated signalling during oocyte in vitro maturation (IVM). Bovine cumulus-oocyte complexes underwent IVM with FSH (FSH+) or without FSH (control/CTL) for 22 h, and CCs were subjected to gene expression profiling. Five software identified reference genes for RT-qPCR (ATP1A1, UBB, and YWHAZ). The transcript levels of FSH-responsive genes HAS2 and PTGS2 (COX2) validated the experimental design. Among candidate TFs, MYC was down-regulated (0.35-fold; P < 0.0001), and THAP11 (RONIN) was up-regulated (1.47-fold; P = 0.016) under FSH+ conditions. In silico analyses predicted binding motifs at MYC and THAP11 genes for previously known FSH-responsive TFs. Signalling pathways (EGFR, ERK, GSK3, PKA, and P38) may execute post-translational regulation due to potential phosphorylation sites in MYC and THAP11 proteins. Prediction of protein–protein interaction networks showed MYC as a core component of FSH signalling, albeit THAP11 acts independently. Hence, MYC integrates FSH signalling networks and may assist in exploring genome-wide transcriptional changes associated with the acquisition of oocyte competence.

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“…Primer design was based on the following criteria: amplicon size (70-200 bp), GC content (40-60%), primer size (19-23 bases), and avoiding primer dimers and hairpin loops (Table 1). Primer sequences were also obtained from previous reports (Moura et al, 2018(Moura et al, , 2019Silva et al, 2018).…”

Section: Primer Designmentioning

confidence: 99%

Housekeeping genes for RT-qPCR in ovine preimplantation embryos

Nascimento

Moura

Silva

et al. 2020

Zygote

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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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Activity of non-canonical pluripotency-associated transcription factors in goat cumulus-oocyte complexes

Cited by 7 publications

References 51 publications

Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set

Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set

MYC integrates FSH signalling networks in cumulus cells during bovine oocyte maturation

Housekeeping genes for RT-qPCR in ovine preimplantation embryos

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