Insights into epigenetic patterns in mammalian early embryos

Mammalian fertilization begins with the fusion of two specialized gametes, followed by major epigenetic remodeling leading to the formation of a totipotent embryo. During the development of the pre-implantation embryo, precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality, but the underlying molecular mechanisms remain elusive. For the past few years, unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development, taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies. The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals, including DNA methylation, histone modifications, chromatin accessibility and 3D chromatin organization.

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106), G2 (2*106) and G3 (4*106) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chi-square (P<0.05) and correlated with CASA and fluorescence data using Person Correlation (P<0.05). The IVF efficiency, cleavage and total blastocyst rates did not show any significant difference (P>0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P<0.05) without difference (P>0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P<0.05), without significant difference (P>0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0.05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106), G2 (2*106) e G3 (4*106) espermatozoides/mL. Às 18h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o Computer-Assisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P<0,05). A eficiência da FIV, taxas de clivagem e blastocisto total não mostraram diferença significativa (P>0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P<0,05), sem diferença (P>0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P<0,05), sem apresentar diferença significativa (P>0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Review of the family Rivulidae (Cyprinodontiformes, Aplocheiloidei) and a molecular and morphological phylogeny of the annual fish genus Austrolebias Costa 1998

The family Rivulidae is the fourth most diverse clade of Neotropical fishes. Together with some genera of the related African family Nothobranchiidae, many rivulids exhibit a characteristic annual life cycle, with diapausing eggs and delayed embryonic development, which allows them to survive in the challenging seasonal ponds that they inhabit. Rivulidae also includes two species known as the only the self-fertilizing vertebrates and some species with internal fertilization. The first goal of this article is to review the systematics of the family considering phylogenetic relationships and synapomorphies of subfamilial clades, thus unifying information that is dispersed throughout the literature. From this revision, it is clear that phylogenetic relationships within Rivulidae are poorly resolved, especially in one of the large clades that compose it, the subfamily Rivulinae, where conflicting hypotheses of relationships of non-annual and annual genera are evident. The second goal of this work is to present an updated phylogenetic hypothesis (based on mitochondrial, nuclear, and morphological information) for one of the most speciose genus of Rivulidae, Austrolebias. Our results confirm the monophyly of the genus and of some subgeneric clades already diagnosed, but propose new relationships among them and their species composition, particularly in the subgenus Acrolebias.

a familia Rivulidae es el cuarto clado más diverso dentro de los peces Neotropicales. Junto con algunos géneros de la familia Nothobranchiidae, muchos rivulidos presentan un característico ciclo de vida anual, con huevos resistentes a la desecación y embriones con diapausas que les permiten sobrevivir en los ambientes estacionales donde habitan. Los Rivulidae presentan también dos especies consideradas como los únicos vertebrados hermafroditas suficientes y algunas especies con inseminación interna. El primer objetivo de este artículo es actualizar la sistemática de la familia considerando las relaciones filogenéticas y las sinapomorfías de los clados que la componen, reuniendo información que se encuentra dispersa en la literatura. De esta revisión surge que las relaciones filogenéticas dentro de Rivulidae están todavía sin resolver, especialmente en uno de los grandes clados que la componen, la subfamilia Rivulinae, donde relaciones conflictivas entre géneros anuales y no anuales son evidentes. El segundo objetivo de este trabajo es presentar una hipótesis filogenética, basada en datos morfológicos, mitocondriales y nucleares, de uno de los géneros más diversos de la familia, el género Austrolebias. Nuestros resultados confirman la monofilia del género y de algunos clados subgenéricos previamente definidos, y propone nuevas relaciones entre ellos, particularmente de las especies del subgénero Acrolebias(AU)

Characterization of HIPSTR highlights the heterogeneous expression pattern of lncRNAs in human embryos and stable cell lines / Caracterização do HIPSTR destaca o padrão de expressão heterogênea de IncRNAs em embriões humanos e linhagens estáveis de células

There is a growing appreciation that eukaryotic genomes are transcribed into numerous, previously undetected - and thus uncharacterized regulatory long non-coding RNAs (lncRNAs). Recent studies are primarily focused on lncRNAs transcribed from intergenic regions and enhancers, leaving antisense lncRNAs the least studied group of lncRNAs. At the same time, antisense transcription occurs in up to 74 % of human gene loci, frequently - from the opposite strand of genes encoding proteins involved in regulation of transcription. Here, we identified HIPSTAR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel conserved lncRNA that is transcribed antisense to the TFAP2A gene. Unlike previously reported antisense lncRNAs, HIPSTR expression does not correlate with the expression of its antisense counterpart. Although HIPSTAR and TFAP2A are co-expressed in in vitro derived neural crest and trophoblast cells, only HIPSTAR and not TFAP2A is specifically expressed in a subset of cells within 8-cell- and morula-stage human embryos. We show that, similar to HIPSTAR, in the individual cells of developing human embryos or of stable cell lines the expression of lncRNAs is more highly heterogeneous than the expression of mRNAs. Finally, we demonstrate that HIPSTAR depletion in HEK293 and H1BP, a human embryonic stem cell line, predominantly affects the expression levels of genes involved in early organismal development and cell differentiation. Together, we show that expression of HIPSTAR and hundreds other lncRNAs is highly heterogeneous in human embryos and cell lines. We use HIPSTAR to exemplify the functional relevance of lncRNAs with heterogeneous and developmental stage-specific expression patterns

Tem sido cada vez mais reconhecido que a transcrição dos genomas eucarióticos produz múltiplos transcritos novos, anteriormente não detectados e ainda não caracterizados, sendo que a maioria é constituida de RNAs não-codificantes longos (lncRNAs) regulatórios. Estudos recentes estão focados principalmente nos lncRNAs transcritos de regiões intergênicas e enhancers; assim, o grupo dos lncRNAs antisenso permanece o menos estudado de todos. Ao mesmo tempo, a transcrição antisenso ocorre em até 74% dos loci de genes humanos, frequentemente - a partir da fita oposta de genes que codificam proteínas envolvidas na regulação da transcrição. No presente trabalho, nós identificamos HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), um lncRNA novo conservado que é transcrito a partir da fita antisenso do gene TFAP2A. Ao contrário do anteriormente relatado para os lncRNAs antisenso, a expressão de HIPSTR não está correlacionada com a expressão do gene da fita oposta. HIPSTR e TFAP2A são co-expressos em células da crista neural e em trofoblastos derivadas in vitro, mas somente HIPSTR e não TFAP2A está especificamente expresso num subconjunto de células de embriões humanos nos estágios de 8-células e mórula. Mostramos que, semelhante a HIPSTR, a expressão de lncRNAs é mais altamente heterogênea que a expressão de mRNAs em células individuais de embriões humanos em desenvolvimento ou em linhagens estáveis de células. Finalmente, nós demonstramos que a depleção de HIPSTAR em células HEK293 e H1BP, uma linhagem de células tronco embrionárias humanas, afeta predominantemente os níveis de genes envolvidos no início do desenvolvimento do organismo e na diferenciação de células. No conjunto, nós mostramos que a expressão de HIPSTR e de centenas de outros lncRNAs é altamente heterogênea em embriões humanos e linhagens celulares. Usamos HIPSTR para exemplificar a relevância funcional de lncRNAs com padrões de expressão heterogêneos e estágio-de-desenvolvimento específicos

Growth differentiation factor 9 and bone morphogenetic protein 15 expression in previtellogenic oocytes and during early embryonic development of Yellow-tail Kingfish Seriola lalandi

BACKGROUND: During fish oocyte maturation, specific molecules are expressed and accumulated within oocyte until fertilization and embryo development. Special attention have been paid in members of the transforming growth factor (TGF-ß) superfamily; growth differentiation factor 9 (GDF9/gdf9) and bone morphogenetic protein 15 (BMP15/bmp15), which exert regulatory functions during oocyte maturation and follicle development. However, little attention has been paid to the involvement of these molecules during embryogenesis considering its importance for the formation of a good quality egg and subsequent embryo survival. The purpose of this study was to analyze the expression of gdf9 andbmp15 in previtellogenic oocytes and during early embryonic development in Seriola lalandi, a pelagic fish with increasing prospect for its aquaculture development, which however, show high mortality at embryo and larval stages. RESULTS: Through RT-qPCR it was found that gdf9 expression was higher in previtellogenic oocytes decreasing after ovulation. This expression profile agrees with its participation in early stages of the follicular development. The transcripts for bmp15 also showed the highest levels in previtellogenic oocytes, however this expression was lower than obtained with gdf9. Conversely, in recently spawned oocytes mRNA bmp15 levels were highest than observed to gdf9. This, is consequent with the main role proposed for this growth factor at the final fish oocyte maturation: avoid the ovulation of an immature oocyte. During embryo development, low levels of mRNA were detected to gdf9, with an increase in 48 H post-fertilization embryos. The bmp15 expression did not change throughout development and was higher than gdf9 at 16 cells, blastula and appearance embryos stages. CONCLUSIONS: Both (gdf9 and bmp15) expression profiles in previtellogenic oocytes and newly spawned eggs are consistent with the described functions for these growth factors in vertebrate ovarian physiology in early and late stages of the follicular development. So, these genes could be considered as quality biomarkers at these stages. However, further studies of these proteins throughout folliculogenesis, are necessaries to fully understand their functions during the oocyte formation. In addition, the persistent expression of these growth factors during development, allows us to speculate possible roles in embryonic processes, which must also be addressed.

Novel insights into the genetic and epigenetic paternal contribution to the human embryo

The integrity of the sperm genome and epigenome are critical for normal embryonic development. The advent of assisted reproductive technology has led to an increased understanding of the role of sperm in fertilization and embryogenesis. During fertilization, the sperm transmits not only nuclear DNA to the oocyte but also activation factor, centrosomes, and a host of messenger RNA and microRNAs. This complex complement of microRNAs and other non-coding RNAs is believed to modify important post-fertilization events. Thus, the health of the sperm genome and epigenome is critical for improving assisted conception rates and the birth of healthy offspring.

RIC-8B, um fator trocador de nucleotídeo guanina (GEF), é essencial para a embriogênese / RIC-8B, a guanine nucleotide exchange factor (GEF), is essential for embryogenesisPalavras-chave em inglês Cilia

RIC-8B é uma proteína que apresenta, in vitro, atividade de fator de troca de nucleotídeos guanina (GEF). No entanto, seu papel in vivo não é conhecido. Dados anteriores do nosso laboratório demonstraram que essa proteína interage especificamente com Gαolf, que é uma proteína G exclusiva do sistema olfatório, presente nos cílios dos neurônios olfatórios, onde ocorre a transdução de sinal ativada pelos odorantes. No camundongo adulto verificou-se, por meio de ensaios de hibridização in situ, que RIC-8B está presente somente em regiões de expressão de Gαolf: no epitélio olfatório maduro e no núcleo estriado do sistema nervoso central. Para avaliar a função fisiológica de RIC-8B in vivo, resolvemos gerar uma linhagem de camundongo knockout para Ric-8B. Verificamos que a linhagem é inviável devido à letalidade dos embriões já em fases precoces do desenvolvimento (por volta de E8,5 e E9,0). A coloração de embriões com X-gal mostra que RIC-8B é especificamente expressa em regiões que darão origem ao sistema nervoso, como na região ventral do tubo neural, e em regiões cefálicas. Interessantemente, mostramos que RIC-8B é expressa na placa do assoalho do tubo neural, de uma maneira muito semelhante ao padrão de expressão de Sonic Hedgehog (SHH), que apresenta um papel fundamental para a organização do sistema nervoso, entre outras funções. Nossos resultados indicam, portanto, que RIC-8B desempenha um papel crucial durante a embriogênese, e que este papel pode estar relacionado com o papel exercido por SHH. Além disso, como a via de sinalização de SHH ocorre em cílios primários nas células alvo, nossos dados levantam a interessante possibilidade de que RIC-8B apresenta funções relacionadas a cílios, tanto no camundongo adulto (neste caso nos cílios dos neurônios olfatórios) como no embrião (neste caso nos cílios primários)

RIC-8B is a protein that, in vitro, acts as a guanine nucleotide exchange factor (GEF). However, its role in vivo remains unknown. Previous data from our laboratory demonstrated that this protein is able to interact specifically with Gαolf, a G protein found only in the olfactory system. This G protein is located in the cilia from olfactory neurons, where odorant signaling occurs. In situ hybridization experiments showed that RIC-8B, in adult mice, is expressed only in regions where Gαolf is expressed, such as the olfactory epithelium and the nucleus striatum in the central nervous system. In order to determine the function of RIC-8B in vivo, we decided to generate a knockout mouse strain for Ric-8B. We found that this strain is not viable due to the lethality of embryos in the early stages of development (around days E8.5 and E9.0). X-gal staining of embryos shows that RIC-8B is specifically expressed in regions that originate the nervous system, such as the ventral neural tube and also cephalic regions. Interestingly, we show that RIC-8B is restrictedly expressed in the floor plate of the neural tube, in a pattern that is very similar to the one shown by Sonic Hedgehog (SHH). The SHH protein plays a fundamental role in the organization of the nervous system, among other functions. Therefore, our results indicate that RIC-8B plays an essential role during the embryogenesis, and that this role can be related to the role played by SHH. Furthermore, because the SHH signaling pathway occurs in primary cilia in the target cells, our data raise the interesting possibility that the role played by RIC-8B is related to ciliary functions, both in adult mice (in this case, in olfactory cilia), and in the embryo (in this case, in primary cilia)

Displasia renal em cão da raça Rotweiller / Renal dysplasia in a dog breed Rotweiller

Descreveram-se os achados clínicos e patológicos de um caso de displasia renal em um cão da raça Rotweiller com oito meses de idade. O animal apresentou vômitos, emagrecimento, polidpsia e poliúria. Houve elevação sanguínea de creatinina, cálcio e da fosfatase alcalina. À necropsia, notaram-se os rins diminuídos de tamanho, com estruturas císticas proeminentes sobre a superfície natural do órgão e, ao corte, firmes e com estruturas císticas distribuídas pelo parênquima. Na avaliação histológica, havia glomérulos imaturos, fibroplasia intersticial e dilatação cística tubular.

In this study we describe the clinical and pathological findings of a case of renal dysplasia in a dog from the Rottweiler breed at 8 months of age. The animal presented vomiting, weight loss, polydipsia and polyuria. There was an increase of blood creatinine, calcium, and alkaline phosphatase. At necropsy it was noted that the kidneys were reduced in size, with prominent cystic structures on the natural surface of the body and the cutting and firm with cystic structures distributed throughout the parenchyma. The histological evaluation was immature glomeruli, interstitial fibroplasia and tubular cystic dilation.

Impronta Genómica y Desarrollo Embrionario / Genomic Imprinting and Embryonic Development

En los organismos diploides, cada gen autosómico está representado por dos copias, o alelos, heredados de cada progenitor al momento de la fecundación. Para la gran mayoría de los genes la expresión ocurre desde ambos alelos de manera simultánea. Sin embargo, un número reducido de genes (menos del 1%) es afectado por un proceso de impronta genómica. Este proceso determina que la expresión del gen sea dependiente del origen parental, es decir, se comporte de manera distinta si su origen es materno o paterno. La metilación del ADN es una de las modificaciones epigenéticas mejor estudiadas y su participación resulta esencial durante el establecimiento de la impronta genómica. Si bien los patrones de metilación a nivel genómico son estables y heredables, existen al menos dos períodos del desarrollo embrionario de mamíferos durante los cuales los patrones de metilación globales son borrados y re-establecidos. Estos dos períodos del desarrollo coinciden con el borrado y establecimiento de la impronta genómica específica de cada individuo. Desde el punto de vista funcional, la mayoría de los genes sometidos a impronta cumplen roles en el control del crecimiento y desarrollo embrionario y placentario. Alteraciones en el patrón de expresión de ellos han sido relacionados a patologías tales como el Síndrome de Algelman y el Síndrome de Prader-Willi, entre otros.

In diploid organisms, autosomal genes are composed of two copies, or alleles, inherited from both parents at fertilization. For the vast majority of autosomal genes, expression occurs from both alleles simultaneously. However, a small proportion (<1%) of genes are imprinted, meaning that their expression depends on the parental origin . DNA methylation is one of the most known epigenetic modifications and its function is critical for the establishment of imprinting. The global pattern of genomic methylation is stable and inheritable, however, it is erased and re-established in a sex-depended manner at two critical periods of embryonic development. Functionally, the majority of imprinted genes play roles in the control of embryonic and placental growth and development. Alterations in imprinted genes have been correlated with several pathologies including the Angelman and Prader-Willi syndromes.

Reproduction of the Brazilian snapper, Lutjanus alexandrei Moura & Lindeman, 2007 (Perciformes: Lutjanidae), off the northern coast of Pernambuco, Brazil

Reproductive aspects of the Brazilian snapper Lutjanus alexandrei, were characterized, including a description of the development of oocytes and spermatogenic cells, size at first sexual maturity, and fecundity. A total of 540 fish were analyzed with 250 having their gonads sectioned to allow microscopic evaluation. Six maturity stages were identified for females and males: immature, maturing, mature, spawning, spawned, and resting. Fish standard length (SL) varied from 13.0 to 28.3 cm and sex ratio was 1.6 males: 1.0 females. Monthly distributions of mean Gonadosomatic Index (GSI) and maturity stages suggest that spawning occurs mainly in a protracted period, during the warmer months, from November to March. The size of first sexual maturity was estimated at 17.1 cm SL for females and 16.8 cm SL for males. Oocyte development suggests that L. alexandrei exhibits a multiple batch spawning behavior and batch fecundity varied from 34,000 to 324,000 oocytes.

Os aspectos reprodutivos da baúna-de-fogo Lutjanus alexandrei foram caracterizados, incluindo a descrição do desenvolvimento dos ovócitos e células espermatogênicas, do tamanho de primeira maturação sexual, e da fecundidade. Um total de 540 peixes foi analisado, dos quais 250 tiveram as suas gônadas seccionadas para avaliação microscópica. Seis estágios de maturidade sexual foram determinados para fêmeas e machos: imaturo, em maturação, maduro, desovando, desovado e repouso. O comprimento padrão (CP) dos peixes variou de 13,0 a 28,3 cm e a proporção sexual foi de 1,6 machos: 1,0 fêmeas. As distribuições mensais dos valores médios do Índice Gonadosomático (IGS) e dos estágios de maturidade sexual sugerem a ocorrência de desovas em um período prolongado, principalmente nos meses de temperaturas mais quentes, entre novembro e março. O tamanho médio de primeira maturação sexual foi estimado em 17,1 cm CP para as fêmeas e 16,9 cm CP para os machos. O padrão de desenvolvimento dos ovócitos sugere que L. alexandrei exibe comportamento de múltiplas desovas por lote, e a fecundidade variou entre 34.000 a 324.000 ovócitos.

Prevalence of bifid tongue and Ankyloglossia in South Indian population with an emphasis on its embryogenesis / Prevalencia de lengua bífida y anquiloglosia en el Sur de la población India con énfasis en su embriogénesis

Disturbance in the organogenesis of tongue might lead to some malformations like tongue tie, bifid tongue and hairy tongue. Severe degrees of these anomalies may cause speech impairment or periodontal defects. The present study was done on patients of the southern coastal belt of India during the past two years, on gross tongue anomalies. The results of the present study reveal that occurrence of tongue tie is 0.2 percent and bifid tongue is 0.3 percent in the southern coastal population. Since great majority of these oral anomalies have genetic basis the purpose of the present report is to highlight that these anomalies can exist without any familial background and also to suggest that environmental factor may play a role in the etiogenesis of these anomalies.

La alteración en la organogénesis de la lengua puede dar lugar a algunas malformaciones como anquiloglosia, lengua bífida y lengua vellosa. Grados severos de estas anomalías puede provocar un trastorno del habla o defectos periodontales. El presente estudio se realizó, durante los últimos dos años, en pacientes de la franja costera del Sur de la India con anomalías graves en la lengua. Los resultados del estudio revelaron que, en la población costera del sur, la incidencia de anquiloglosia era de 0,2 porciento y de lengua bífida de 0,3 por ciento. Dado que la gran mayoría de estas anomalías orales tienen base genética, el propósito del presente informe fue poner de relieve que estas anomalías pueden existir sin ningún tipo de antecedentes familiares y también sugerir que los factores ambientales podrían jugar un papel en el etiogenesis de estas anomalías.

Regeneración vía embriogénesis somática de una variedad colombiana élite de Theobroma cacao L / Regeneration through somatic embryogenesis of an elite colombian Theobroma cacao L variety

El objetivo de esta investigación fue evaluar dos protocolos de propagación vía embriogénesis somática a partir de explantes florales en dos clones élite BIOB e ICS95 de Theobroma cacao L. Se obtuvo un 50 y 32% de callo embriogénico en ICS95 y BIOB respectivamente con el protocolo de Fontanel et al. (2002), modificado después de un periodo de cultivo de tres meses. Los embriones pasaron por fases que se correspondieron con medios de cultivo diferenciales: Inducción, Formación, Maduración y Mantenimiento. Para la embriogénesis somática secundaria se obtuvo un 23% de embriones a partir de embriones somáticos primarios en un medio, conteniendo 1mg/L de 2,4,5 T (2,4,5 Triclorofenoxiacético). Se logró, además, desarrollar enraizamiento adventicio aplicando pulsos de IBA (Ácido Indol Butírico) a 0.5mg/L y 0.5g/L durante un minuto. Las plantas enraizadas se llevaron a una mezcla de tierra: arena (1:1) para su adaptación ex vitro, obteniéndose un 66% de plantas aclimatadas. Los estudios histológicos mostraron diferentes características típicas del desarrollo embriogénico. Este es el primer reporte en el que se logra de manera exitosa la conversión hasta plántula (68%) y la adaptación ex vitro de una variedad colombiana de cacao vía embriogénesis somática primaria y secundaria.

In this research we evaluate two protocols of propagation via somatic embryogenesis from floral explants using two elite clones BIOB and ICS95 of Theobroma cacao L. We obtained 50 and 32% of embryogenic callus on ICS95 and BIOB respectively with Fontanel et al., (2002) protocol modified after three months of culture. The embryos went through four phases; Induction, Formation, Maduration and Mantenimiento which corresponded each one with different media culture. For secondary somatic embryogenesis we obtained 23% of embryos from primary somatic embryos in a medium with 1mg/L of 2,4,5 T (2,4,5 Triclorofenoxiacetic). Also we obtained plants that developed new roots applying pulses with IBA (Indol Butiric Acid) 0.5mg/L and 0.5g/L for a minute. The developed plants were moved to a mix of potting soil and sand (1:1) for their ex vitro adaptation, getting 66% of acclimatized plants. The histological analysis showed the typical characteristics of the embryogenic development. This is the first report where it is achieved the successful conversion to plantlets (68%) and ex vitro adaptation of a colombian cocoa variety via primary and secondary embryogenesis.

Desenvolvimento de embriões de camundongas após manutenção em diferentes soluções de manipulação / Development of mice embryos after maintenance in different manipulation solutions

Avaliou-se a eficácia de duas soluções de manipulação (SM) de embriões de camundongas nos estádios de blastocisto inicial (Bin), mórula compacta grau I (McI) e II (McII), distribuídos aleatoriamente em três tratamentos (T), de acordo com a solução de manutenção. No T1 usou-se PBS modificado (controle); no T2, SME e no T3, SME enriquecida. Os embriões foram mantidos durante quatro horas na solução de manutenção e posteriormente classificados quanto ao estádio de desenvolvimento e à qualidade embrionária. Logo após, foram cultivados em meio TCM 199 e classificados novamente quanto ao estádio de desenvolvimento e à qualidade embrionária. A taxa de desenvolvimento dos embriões após manutenção por quatro horas em solução de manipulação foi menor (P<0,05) nos embriões do controle, comparada à de embriões do SME e SME enriquecida, diferença esta não observada (P>0,05) após o cultivo in vitro. Os embriões McII do T3 tiveram maior desenvolvimento (P<0,05) em relação aos embriões do T1 e T2, indicando o efeito benéfico do enriquecimento da solução SME. Conclui-se que as soluções de manipulação SME e SME enriquecida influenciaram beneficamente o desenvolvimento de embriões.

The effect of embryo manipulation solution followed by in vitro culture in mice embryos was studied. The embryos at early blastocyst (Bin), and compact morula grades I (McI) and II (McII) were randomly assigned into three treatments. T1 used modified PBS (control), T2 used EMS, and T3 used EMS supplemented. In each treatment, the embryos were kept in manipulation solution for four hours. Finishing the manipulation period, the embryos were classified according the development stage and quality. Following, the embryos were cultured in TCM 199. After the culture period, the embryos were evaluated according to quality and development stage. The development rate for Bin, McI, and McII after maintenance for four hours in manipulation solution was lower for control embryo (P>0.05) as compared to EMS and EMS supplemented embryos. After in vitro culture, no differences (P>0.05) on embryo development rate among control, EMS, and EMS supplemented were observed. Moreover, McII from EMS supplemented had a higher development (P<0.05) (93 percent) as compared to control (82.5 percent) and EMS (83.9 percent), suggesting a beneficial effect of EMS supplemented. EMS and EMS supplemented embryos had a positive effect on embryo development, showing higher embryo development than those in PBS solution.

Selection of reference genes for normalization of quantitative real-time PCR in cell cultures of Cyclamen persicum

As a prerequisite for gene expression analyses in cell cultures of the ornamental crop Cyclamen persicum basic parameters for quantitative real-time polymerase chain reaction (qRT-PCR) have been established including the selection of reference genes using the software tools ‘geNorm’ and ‘NormFinder’. Five potential reference genes have been tested (elongation factor tu (Ef-Tu), putative ABC transporter ATPase, putative conserved oligomeric Golgi (COG) complex component, V-ATPase G subunit 1 and Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4)). ‘NormFinder’ as well as ‘geNorm’ identified Ef-Tu to be the least stable reference gene while the ranking of the most stable genes differed depending on the algorithm. According to ‘NormFinder’ COG complex component displayed the most stable expression whereas ‘geNorm’ indicated V-ATPase G subunit 1 and a putative ABC transporter ATPase to be the most reliable reference genes. Hence, we concluded to use a normalization factor calculated from the four reference genes V-ATPase G subunit 1, ABC transporter ATPase, Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4) and COG complex component for normalization of qRT-PCR in cell cultures of Cyclamen persicum.

Somatic development and embryo yield in crossbred F1 mice generated by different mating strategies / Desenvolvimento somático e produção de embriões em camundongos cruzados F1 gerados com diferentes estratégias de cruzamento

The aim of this study was to evaluate different mating strategies among endogamic strains to create F1 populations of mice, minimising the effect of inbreeding depression on somatic development and embryo yield. Females from the strains Swiss, CBA and C57Bl/6 were divided in nine experimental mate arrangements. The total numbers of pups born alive per dam and somatic development, estimated by weighing and measuring the crown-rump length, were recorded. Superovulation response was evaluated in outbreed females. Litter size differed among endogamic dams, irrespective of the sire. Somatic development results suggest heterosis and imprinting phenomena, once a differential parental effect was demonstrated. There was no difference in corpora lutea, ova or embryos recovered (P > 0.05), but recovery and viability rates differ among F1 groups (P < 0.05). The association of dam prolificity with somatic development and superovulation response of the pups should be considered for experimental F1 populations establishment. The use of outbreed animals, however, did not reduce response variability to hormone treatment.

Objetivou-se neste estudo avaliar diferentes estratégias de cruzamento entre linhagens endogâmicas para a formação de populações de camundongos F1, minimizando o efeito da depressão por endogamia nos resultados de desenvolvimento somático e produção de embriões. Fêmeas das linhagens Swiss, CBA e C57Bl/6, foram distribuídas em nove possíveis cruzamentos. Foram registrados o número de filhotes nascidos vivos por matriz e o desenvolvimento somático dos mesmos, mensurado pelo peso e comprimento. A resposta superovulatória foi avaliada nas fêmeas cruzadas. O tamanho das ninhadas diferiu entre as linhagens das matrizes, de forma independente da linhagem dos reprodutores. Os resultados do desenvolvimento somático sugerem a ocorrência de heterose e imprinting, uma vez que foi demonstrado um efeito parental diferenciado. Não foram observadas diferenças no número de corpos lúteos, estruturas ou embriões recuperados (P > 0,05), mas as taxas de recuperação e o percentual de embriões viáveis diferiram entre os grupos (P < 0,05). A associação da prolificidade da linhagem das matrizes com as características do desenvolvimento somático e resposta superovulatória dos filhotes deve ser considerada no estabelecimento de populações experimentais F1. O uso de animais cruzados, contudo, não reduziu a variabilidade da resposta aos tratamentos hormonais.

Normalized embryoid cDNA library of oil palm (Elaeis guineensis) / Biblioteca ADNc normalizada de embrioides en aceite de palma (Elaeis guineensis)

A normalized embryoid cDNA library (EON) was constructed based on reassociation kinetics reaction. Results from dot blot hybridization and sequencing of EON cDNA clones clearly indicated that the normalization process reduced the frequency of high abundance transcripts and increased the frequency of low abundance gene transcripts. A total of 553 non-redundant expressed sequence tags (ESTs) were identified, 325 of these were not observed in the standard oil palm cDNA libraries sequenced previously. A total of 10 EON cDNA clones were chosen for expression profiling across samples from different stages of the tissue culture process. Two of the genes exhibited promising expression patterns for predicting the embryogenic potential in callus. Some of these genes were also differentially expressed in the various tissues of oil palm. This study showed that normalization of the existing embryoid library improved the chances of identifying transcripts not captured in the standard libraries, some of which could be associated with embryogenesis. This collection of ESTs is particularly well suited for use as candidate genes for development of an oil palm DNA chip, which can be used to obtain a more comprehensive view of the molecular mechanism associated with oil palm tissue culture.

Induction of somatic embryogenesis as an example of stress-related plant reactions / Inducción de embriogénesis somática como un ejemplo de las reacciones de la planta en relación al estres

In this review, we address the role of stress as one of the principal causes for a cell or tissue to change its pre-existing somatic program, reprogramming itself to express the embryogenic pathway. The focus of this paper is the effect of different stress conditions on the induction phase of plant somatic embryogenesis, as well as the development of embryogenic competence as a result of the applied stresses. We also present a variety of data that link plant somatic embryogenesis, DNA methylation and oxidative stress response.

Efecto de la densidad de inóculo en la multiplicación y diferenciación de suspensiones celulares embriogénicas en el cultivar híbrido de banano FHIA-18 (Musa spp AAAB) / The effect of inoculum density on embryogenic cell suspension proliferation and differentiation in banana hybrid cultivar FHIA-18 (Musa spp AAAB)

La obtención de un sistema de regeneración eficiente por medio de la embriogénesis somática en las Musaceas, es hoy una gran herramienta ante los enormes problemas que presenta este género con el ataque de enfermedades como la sigatoka negra. El objetivo del trabajo es determinar las densidades celulares adecuadas para las etapas de multiplicación de suspensiones celulares embriogénicas y formación de los embriones somáticos en medios de cultivo líquidos. Como material vegetal se usaron brotes inmaduros de la inflorescencia masculina de Musa AAAB, cv. FHIA-18. Los resultados demostraron que es posible el establecimiento de suspensiones celulares homogéneas a partir de embriones somáticos en etapa globular, y obtener los mayores volúmenes de biomasa celular al multiplicar dichas suspensionescon una densidad del 3% del volumen de células sedimentadas. A partir del decimoquinto día en el medio de cultivo de formación de embriones comenzaron a formarse estructuras compuestas por proembriones y embriones somáticos en etapa globular; entre las densidades estudiadas los mejores resultados se obtuvieron con 100 mgMF en la cual se formaron 1 871 ES.l-1 con un peso de 248 mgMF.l-1.

An extremely useful tool for dealing with the enormous problems involved in banana growing (Musaceae) caused by the attack of diseases such as black Sigatoka can be obtained today by ensuring an efficient regeneobration system via somatic embryogenesis. The work was aimed at defining appropriate cell densities for embryogenic cell suspension growth stages and somatic embryo formation in liquid culture medium. Immature male inflorescence buds from Musa AAAB cf FHIA-18 were used as vegetal material. The results showed that it is possible to establish homogeneous cell suspensions from somatic embryos in globular stage andobtain greater cell biomass volume by multiplying the suspension with 3% sedimented cell volume (density).Embryos began to form structures in culture medium consisting of globular stage somatic proembryos and embryos from the fifteenth day onwards. The best results amongst the densities studied were obtained with 100 mgMF, in which 1,871 ES.l -1 were formed weighing 248 mgMF.l-1.

Establishment of an efficient callus induction and plant regeneration system in Pakistani wheat (Triticum aestivum) cultivars

Four commercially grown wheat varieties of Pakistan, namely Inqilab-91, Chakwal-97, Tatara and Manthar were used for this investigation. For callus induction different concentrations of 2,4-Dichlorophenoxyaceticacid (2,4-D) along with 0.1 mg/L of Kinetin were evaluated. For regeneration initially different concentrations of Indole-3-Acetic Acid (IAA) and 6-BenzylAminoPurine (BAP) were tested. Best hormone combinations were further subjected to Kinetin and 6-ã-ã-dimethylallylaminopurine (2iP). For Inqilab-91, Chakwal-97 and Manthar, 3 mg/L of 2,4-D was found optimum, which induced 83.25 percent, 77.75 percent and 95.20 percent of embryogenic calli, respectively. Maximum callus induction (97.18 percent) was observed in Tatara when 2 mg/L of 2,4-D was used. As regard to regeneration, Inqilab-91, Chakwal-97 and Manthar showed maximum regeneration on media containing 0.1 mg/L IAA, 0.4 mg/L Kinetin and 0.5 mg/L 2iP, regenerating 87.25 percent, 81.75 percent and 68.75 percent respectively. For Tatara maximum regeneration of 12.25 percent was obtained on 0.1 mg/L IAA and 2 mg/L of BAP. Presently optimized regeneration method holds promise for facilitating the deployment of agronomical important trait through genetic transformation for the improvement of this important food crop.

Anatomical study of the horseshoe kidney / Estudio anatómico del riñón en herradura

We present the anatomical study of a horseshoe kidney found during dissection practice at the human anatomy laboratory. The specimen consisted of a renal mass joined at its lower poles by an isthmus composed of renal parenchyma. We provide a macroscopic description of renal blood supply and the excretory system. We also discuss the anatomic and embryologic importance of this anomaly.

Se presenta el estudio anatómico de un riñón en herradura encontrado durante la práctica de disección en el Laboratorio de Anatomía Humana. La muestra consistió de una masa renal unida a sus polos inferiores por medio de un istmo de parénquima renal. Proporcionamos una descripción macroscópica de suministro sanguíneo renal y el sistema excretor. Asimismo, se discute la importancia anatómica y embriológica de esta anomalía.