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

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.

Evolutionary-driven C-MYC gene expression in mammalian fibroblasts.

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), RXRß, 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.

Evaluation of embryo collection and transfer days on pregnancy rate of Mangalarga Marchador mares during the breeding season / 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

Abstract Background: Proper timing for embryo collection and transfer in horses -which is critical for the success of this biotechnology- is still debated. Additionally, there is little information on this technology under tropical conditions. Objective: To determine the best day for collection and transfer of embryos in Mangalarga Marchador mares under Brazilian northeast's conditions. Methods: Donors (n= 30) and recipients (n= 76) in diestrus phase were selected based on both clinical and gynecology examinations. Estrus was induced on both donor and recipient mares by intramuscular injection of 5 mg Dinoprost, aiming to obtain an ovulation interval of -1 to +3 between recipient and donor. Ovulation was induced with buserelin acetate when the largest follicle reached at least 35 mm in diameter. At this time, mares were subjected to artificial insemination at 48-hour intervals until ovulation. The embryos were collected on days 7, 8, and 9 after ovulation. Results: The embryo collection on day 8 was more efficient (p<0.05) than on day 7, but it was not more effective (p>0.05) than day 9, which presented the same efficiency (p>0.05) as day 7. From a total of 76 embryos transferred to the recipients, that were between days 4 and 9 after ovulation, there was no influence (p>0.05) of the day of transfer on pregnancy rate. Conclusions: The embryo collection must be performed on day 8 after ovulation, and transfer can be performed on any day of that interval (4-9) without affecting the pregnancy rate.

Resumen Antecedentes: El momento mas apropiado para la recolección y transferencia de embriones en equinos -que es fundamental para el éxito de esta biotecnología- continua siendo sujeto de estudio. Además, es escasa la información sobre esta tecnología en condiciones tropicales. Objetivo: Determinar el momento mas adecuado para la recolecta y transferencia de embriones en yeguas Mangalarga Marchador, en las condiciones del nordeste Brasileño. Métodos: Donadoras (n= 30) y receptoras (n= 76) en la fase de diestro se seleccionaron con base en los exámenes clínicos y ginecológicos. El estro de las yeguas donadoras y receptoras fue inducido con 5 mg de Dinoprost, vía intramuscular, intentando obtener un intervalo de ovulación de -1 a +3 entre la receptora y la donadora. La ovulación fue inducida con acetato de buserelina cuando el folículo mayor alcanzó 35 mm de diámetro. En ese momento, las yeguas fueron sometidas a inseminación artificial en intervalos de 48 horas hasta la ovulación. Los embriones fueron recolectados en los días 7, 8 y 9 después de la ovulación. Resultados: La recolecta de embriones en el día 8 fue más eficiente (p<0,05) que en el día 7, pero no fue más efectivo (p>0,05) que en el día 9, el cuál presentó la misma eficiencia (p>0,05) que en el día 7. De un total de 76 embriones transferidos a las receptoras, que se encontraban entre el día 4 y 9 después de la ovulación, no se registró influencia (p>0,05) del día de la transferencia en la tasa de preñez. Conclusiones: La recolecta embrionaria debe ser realizada el día 8 después de la ovulación, y la transferencia puede ser realizada en cualquier día de este intervalo (4 a 9) sin que se afecte la tasa de preñez.

Resumo Antecedentes: A importância do momentoda colheita e da transferência do embrião equino para o sucesso dessa biotécnica em equino continua sem ser completamente entendida. Adicionalmente, existe pouca informação sobre essa tecnologia em condições tropicais. Objetivo: Determinar o melhor dia para colheita e para transferência de embriões em eguas manga larga marchador nas condições do nordeste brasileiro. Métodos: Doadoras (n = 30) e receptoras (n = 76) na fase de diestro foram selecionadas com base nos exames clínico e ginecológicos. O estro das éguas doadoras e receptoras foi induzido com 5 mg de Dinoprost administrado por via intramuscular, buscando obter um intervalo de ovulação de -1 a +3 entre a receptora e a doadora. A ovulação foi induzida com acetato de buserelina quando o foliculo maior alcançou o tamanho de 35 mm de diâmetro. Nesse momento, as éguas foram submetidas a inseminação artificial em intervalos de 48 horas até a ovulação. Os embriões foram colhidos nos dias 7, 8 e 9 depois da ovulação. Resultados: A colheita de embriões no dia 8 foi mais eficiente (p<0,05) do que no dia 7, porem não foi mais efetivo (p>0,05) do que o dia 9, o qual apresentou a mesma eficiência (p>0,05) que o dia 7. De um total de 76 embriões transferidos para as receptoras que se encontravam entre os dias 4 e 9 depois da ovulação, não se registrou influência (p>0,05) do dia da transferência sobre a taxa de prenhez. Conclusões: A colheita embrionária deve ser realizada no dia 8 depois da ovulação, e a transferência pode ser realizada em qualquer dia desse intervalo (4-9) sem que a taxa de prenhez seja afetada.

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

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.