ABSTRACT
Infertility or subfertility in bovine males may be related to spermatic microRNAs (miRNAs), whose function seems to be associated with the regulation of gene expression, degradation orstorage of messenger RNAs (mRNAs) for later translation into early embryonic development. Thus, the purpose of this study was to identify differentially expressed miRNAs in semen samples from bulls (Bos taurus) with low and high efficiency in the in vitro embryo production (IVEP) and to evaluate if they can be used as markers of semen efficiency for IVEPs. In order to identify miRNA markers of semen efficiency in thein vitro embryo production, eight semen samples from each animal, one bull with high and two bulls with low efficiency in IVEPs were used to perform the RNAseq technique for miRNAs. Initially the samples were washed with PBS to remove the extender semen and subsequently were submitted to RNA extraction protocols performed according to procedures described by mirVana™ miRNA Isolation Kit. Then, the amplification of the miRNAs was carried out, not to mention the preparation of the library (Ion Total RNA-Seq Kit v2), the PCR emulsion reaction, enrichment, as well as the injection of the sample on the chip by the Ion Chef equipment. The sequencing was done on Ion Proton equipment. The comparison between the samples was established using two methodologies for searching for targets to increase the robustness of the analytical procedure: the miRanda program using as cutoff minimum free energy of the hybridization -20 kcal/Mol, 100% of identity between nucleotides 2 and 8 of the miRNA, and the RNAhybrid program, using as cutoff minimum free energy of hybridization -20 kcal/mol. In sum, 1306 miRNAs were identified in the samples. The bta-miR-380-5p, bta-miR-155, bta-miR-30c and bta-miR-34a genes were identified by the Bioinformatics as being strongly differentially expressed between the groups, indicating that these genes may present themselves as possible efficiency markers. However, it has become clear that there is no single miRNA that marks different types and causes of fertility problems.
A infertilidade ou subfertilidade em machos bovinos pode estar relacionada a microRNAs espermáticos (miRNAs), cuja função parece estar associada à regulação da expressão gênica, degradação ou armazenamento de RNAs mensageiros (mRNAs), para posterior tradução no desenvolvimento embrionário inicial. Assim, o objetivo deste estudo foi identificar miRNAs diferencialmente expressos em amostras de sêmen de touros (Bos taurus) com baixa e alta eficiência na produção in vitro de embriões (PIVE) e avaliar se eles podem ser utilizados como marcadores de eficiência do sêmen em PIVEs. Para identificar miRNA marcadores da eficiência de sêmen em PIVE, oito amostras de sêmen de cada animal, sendo um touro com alto e dois touros com baixa eficiência, foram utilizados para realizar a técnica de RNAseq para miRNAs. Inicialmente as amostras foram lavadas com PBS para remover o diluente do sêmen e, posteriormente, foram submetidas a protocolos de extração de RNA realizados de acordo com os procedimentos descritos pelo Kit de isolamento de miRNA mirVana ™. Em seguida, foi realizada a amplificação dos miRNAs, a preparação da biblioteca (Ion RNA-Seq Kit v2), a reação de emulsão de PCR, enriquecimento e a injeção das amostras no chip apropriado utilizando o equipamento Ion. Chef. O sequenciamento foi realizado no equipamento Ion Proton. A comparação entre as amostras foi estabelecida utilizando duas metodologias de busca de alvos para aumentar a robustez do procedimento analítico: o programa miRanda utilizando como valor de corte a energia mínima livre de hibridização -20 kcal / Mol e 100% de identidade entre os nucleotídeos 2 e 8 do miRNA, e o programa RNAhybrid, utilizando como valor de corte a energia mínima livre de hibridização -20 kcal / mol. Em suma, 1306 miRNAs foram identificados nas amostras. Os genes bta-miR-380-5p, bta-miR-155, bta-miR-30c e bta-miR-34a foram identificados pela bioinformática como sendo fortemente diferencialmente expressos entre os grupos, indicando que esses genes podem se apresentar como possíveis marcadores de eficiência. No entanto, ficou claro que não existe um único miRNA que marque diferentes tipos e causas de problemas de fertilidade.
Subject(s)
RNA , Cattle , Embryo Research , InfertilityABSTRACT
Changes in hormonal levels can produce alternative phenotypes. Juvenile hormone III plays an importantrole in the regulation of metamorphosis, caste determination and age in bees. In this work, we examined theultrastructure of corpora allata cells from stingless bees (Melipona quadrifasciata) treated with juvenilehormone during development. The corpora allata cells of M. quadrifasciata queens showed greater activitythan those of workers. The topical application of juvenile hormone III altered the cellular ultrastructureand either delayed development (as shown by fewer mitochondria and greater chromatin condensation) orenhanced development (looser chromatin and numerous mitochondria) when compared to untreated (control)bees. Our results show that corpora allata cells differ in their ultrastructural characteristics and that thecessation of juvenile hormone production by these cells in M. quadrifasciata is not synchronous.
Subject(s)
Animals , Bees , Corpora Allata , Corpora Allata/anatomy & histology , Insect Hormones , Hormones/analysis , Hormones/physiologyABSTRACT
The objective of the present study was to determine by differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) the effects of juvenile hormone (JH) III applied during the late larval 3 (L3) phase on gene expression in Melipona scutellaris. A temporal window of expression of feminizing genes exists during the late L3 and pre-defecating larval phases when these genes can be turned on or off by the action of JH, which is able to mediate the differentiation of female larvae into queens. Combination of the HT11A-AP4 primers revealed differential expression in L3 individuals treated with JH III for 1 h, with weak expression of the transcript, while intense expression was observed for controls and individuals treated for 4 h. Combination of the HT11G-AP4 and HT11G-AP5 primers showed suppression of the gene products for each primer combination in 1-h treated larvae compared to untreated control individuals of the same age and individuals treated for 4 h. Differential gene expression was also observed during development. These results demonstrate that the JH III may suppress or alter gene expression profiles during phase L3 of M. scutellaris.