Hardening of zona pellucida of mouse oocytes and embryos in vivo and in vitro

Objective - To evaluate the effect of in vitro culture on zona pellucida resistance in mouse oocytes and embryos. Method-Zona pellucida resistance was assessed by comparing duration of zona lysis in the presence of alpha- chymotrypsin. The effects of artificial or physiological conditions of development were evaluated by comparing embryos in vitro with those left to reach the same stage of development in vivo. Results - The time required for zona lysis of oocytes increased after 2, 9.4, and 48 hours in vitro (P < .001). The same observation holds true for oocytes left in vivo during 24 hours. Fertilization both in vivo and in vitro induced a major increase in zona resistance. At the two-cell stage, in vitro culture did not harden the zona pellucida. At the morula stage and beyond, enzymatic lysis was slightly longer in vitro as compared to that of similar stages recovered from the genital tract. Conclusions - Our data indicate that in vitro culture conditions do not modify zona hardening in oocytes and only slightly increased zona resistance from the morula stage on.

Isolation, culture et caractérisation phénotypique de cellules musculaires lisses endobronchiques équines.

Le muscle lisse endobronchique est l’un des acteurs principaux de l’asthme. La description de ces caractéristiques phénotypiques reste cependant très elliptique, notamment à cause de la difficulté inhérente à l’échantillonnage. Le cheval offre un large champ d’investigation en raison de sa taille et un modèle d’asthme pertinent en regard de la similitude entre asthme et souffle. La technique de culture et de caractérisation du muscle lisse a été mise au point à partir de muscle lisse trachéal. Ce modèle a ensuite été transposé et réalisé à partir de biopsies endobronchiques chez le cheval. Les cellules du muscle lisse ont été isolées, mises en culture puis caractérisées par immunofluorescence, cytométrie de flux et immunobuvardage. Le maintien du phénotype contractile en culture restant un défi dans l’établissement d’un modèle d’asthme réaliste. Suite à l’isolement des cellules musculaires lisses à partir de muscle lisse trachéal équin et leur mise en culture en présence de 10% de FBS pendant 7 passages, 96.4% des cellules expriment l’α-smooth muscle-actine (α-sm-actine), tandis que 83.8% et 77% expriment la desmine et la myosine respectivement. Les cellules musculaires lisses issues de biopsies endobronchiques expriment après 7 passages à 84% l’α-sm-actine, à 57% la desmine et 69% la myosine. Ces résultats ont été obtenus par immunofluorescence et immunobuvardage. Le pourcentage de cellules exprimant les protéines d’intérêt, tout comme l’intensité moyenne de fluorescence ne présentent pas de variation significative ni entre le 4ième et le 7ième passage, ni avec la caractérisation initiale, lors du premier passage. Cette étude suggère qu’il est possible de maintenir le phénotype contractile en culture sur plastique en présence de 10% de FBS, et que les biopsies endobronchiques sont un support d’étude valable pour de futures investigations concernant le rôle du muscle lisse et ses caractéristiques.

Overexpression of coconut AINTEGUMENTA-like gene, CnANT, promotes in vitro regeneration in transgenic Arabidopsis

Knowledge of the molecular biological changes underlying the process of embryogenesis is important for the improvement of somatic embryogenesis of coconut. Among the transcription factors that control the transition from vegetative to embryogenic growth, members of APETALA2/Ethylene-responsive element binding protein domain family play an important role in promoting embryo development. Significant insights into the role of AP2 genes have been obtained by the ectopic expression of AP2 sub family genes in transgenic Arabidopsis. A homolog of the AINTEGUMENTA-like gene that encodes the two AP2 domains and the linker region was identified in the coconut genome. Phylogenetic analysis showed that this gene, CnANT, encodes a protein that branched with BABY BOOM/PLETHORA clade in the AINTEGUMENTA-like major clade and was similar to the oil palm EgAP2-1 protein. According to real time RT-PCR results, higher expression of CnANT was observed in more mature zygotic embryos. Also, high CnANT expression was recorded in embryogenic callus compared to other stages of somatic embryogenesis. We examined the effect of ectopic CnANT expression on the development and regenerative capacity of transgenic Arabidopsis. Overexpression of CnANT in Arabidopsis induced hormone free regeneration of explants. Furthermore, ectopic expression of CnANT enhanced regeneration in vitro and suggested a role for this gene in cell proliferation during in vitro culture.

Global scale transcriptome analysis of Arabidopsis embryogenesis in vitro

Background Somatic embryogenesis (SE) in plants is a process by which embryos are generated directly from somatic cells, rather than from the fused products of male and female gametes. Despite the detailed expression analysis of several somatic-to-embryonic marker genes, a comprehensive understanding of SE at a molecular level is still lacking. The present study was designed to generate high resolution transcriptome datasets for early SE providing the way for future research to understand the underlying molecular mechanisms that regulate this process. We sequenced Arabidopsis thaliana somatic embryos collected from three distinct developmental time-points (5, 10 and 15 d after in vitro culture) using the Illumina HiSeq 2000 platform. Results This study yielded a total of 426,001,826 sequence reads mapped to 26,520 genes in the A. thaliana reference genome. Analysis of embryonic cultures after 5 and 10 d showed differential expression of 1,195 genes; these included 778 genes that were more highly expressed after 5 d as compared to 10 d. Moreover, 1,718 genes were differentially expressed in embryonic cultures between 10 and 15 d. Our data also showed at least eight different expression patterns during early SE; the majority of genes are transcriptionally more active in embryos after 5 d. Comparison of transcriptomes derived from somatic embryos and leaf tissues revealed that at least 4,951 genes are transcriptionally more active in embryos than in the leaf; increased expression of genes involved in DNA cytosine methylation and histone deacetylation were noted in embryogenic tissues. In silico expression analysis based on microarray data found that approximately 5% of these genes are transcriptionally more active in somatic embryos than in actively dividing callus and non-dividing leaf tissues. Moreover, this identified 49 genes expressed at a higher level in somatic embryos than in other tissues. This included several genes with unknown function, as well as others related to oxidative and osmotic stress, and auxin signalling. Conclusions The transcriptome information provided here will form the foundation for future research on genetic and epigenetic control of plant embryogenesis at a molecular level. In follow-up studies, these data could be used to construct a regulatory network for SE; the genes more highly expressed in somatic embryos than in vegetative tissues can be considered as potential candidates to validate these networks.

In vitro Survival of Follicles Collected from Domestic Cats' Ovaries at Different Stages of Oestrous Cycle and Cultured with IGF-1

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The effects of ovalbumin as a protein source during the in vitro production of bovine embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crescimento in vitro e aclimatização de Cattleya loddigesii Lindley (Orchidaceae) com carvão ativado sob dois espectros luminosos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phosphatidylcholine and Sphingomyelin Profiles Vary in Bos taurus indicus and Bos taurus taurus In Vitro- and In Vivo-Produced Blastocysts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito da concentração de ácido 3-indol-acético na ativação e crescimento in vitro de folículos pré-antrais ovinos

Avaliou-se o desenvolvimento de folículos pré-antrais ovinos após o cultivo in vitro do córtex ovariano em várias concentrações de ácido 3-indol acético (IAA). O córtex ovariano foi dividido em fragmentos de aproximadamente 3×3mm. Um fragmento foi imediatamente fixado em Bouin (controle - dia 0) e os demais destinados ao cultivo por dois ou seis dias em meio essencial mínimo (MEM+) acrescido de 10, 40, 100, 500 ou 1000ng/ml de IAA. Após o cultivo in vitro, não houve variação entre folículos dos tratamentos e folículos-controle, exceto nos suplementados com 40ng/ml de IAA. Nestes observaram-se redução de folículos primordiais e aumento de folículos em desenvolvimento (P<0,05). em relação aos folículos do grupo-controle, houve redução de pré-antrais normais no cultivo de seis dias (P<0,05). Após dois dias de cultivo, a redução foi observada somente nos folículos suplementados com 500 ou 1000ng/ml de IAA. Folículos pré-antrais ovinos podem ser ativados in vitro com sucesso após o cultivo em MEM+ suplementado com 40ng/ml de IAA.

Avaliação do efeito das concentrações de sacarose e dos estádios de desenvolvimento do fruto no cultivo in vitro de embriões de frutos de cafeeiro

A cultura in vitro é uma técnica controlada que proporciona estudar os processos nutricionais, fisiológicos e bioquímicos de embriões em vários estádios de desenvolvimento. O objetivo foi avaliar a relação entre estádio de desenvolvimento do fruto e concentração de sacarose, no meio, durante o desenvolvimento, in vitro, de embriões de cafeeiro. Frutos de Coffea arabica cv. Acaiá foram colhidos, lavados, desinfestados, seus embriões excisados e inoculados em meio de cultivo MS, com pH ajustado para 5,8. Os tratamentos consistiram em combinação de concentrações de sacarose (0, 15, 30, 60, 90 e 120 g L-1) e estádios de desenvolvimento do fruto (chumbinho, chumbo, verde, verde-cana, cereja e passa). Após a inoculação, os embriões foram incubados em sala de crescimento, a 27 ± 1 ºC, fotoperíodo de 16 horas e 35 µ mol m-2 s-1 de intensidade luminosa. O delineamento experimental utilizado foi inteiramente casualizado, em esquema fatorial 6 x 6, com seis repetições constituídas por quatro tubos cada. Após 60 dias de desenvolvimento, as plântulas foram avaliadas com base no comprimento da parte aérea, massa de matéria fresca total da parte aérea e das raízes. Observaram-se influências das concentrações de sacarose e dos estádios de desenvolvimento do fruto no crescimento e desenvolvimento das plântulas. Resultados satisfatórios para todas as variáveis estudadas foram obtidos com embriões excisados no estádio verde, inoculados em meio de cultivo suplementado com 51 a 70 g L-1 de sacarose.

Interactions of indole acetic acid with EGF and FSH in the culture of ovine preantral follicles

The mechanisms that regulate the gradual exit of ovarian follicles from the non-growing, primordial pool are very poorly understood. The objective of this study was to evaluate the effects of adding indole acetic acid (IAA), epidermal growth factor (EGF) and follicle stimulating hormone (FSH) to the media for in vitro culture of ovine ovarian fragments and determine their effects on growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2 or 6 days in culture plates with: minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, bovine serum albumine and antibiotics (MEM+); MEM+ plus IAA (40 ng/mL); MEM+ plus EGF (100 ng/mL); MEM+ plus FSH (100 ng/mL); MEM+ plus IAA + EGF; MEM+ plus IAA + FSH; MEM+ plus EGF + FSH; or MEM+ plus IAA + EGF + FSH. After 2 or 6 days of culture in each treatment, the pieces of ovarian cortex were fixed in Bonin for histological examination. Follicles were classified as primordial or developing (primary and secondary) follicles. Compared to the control, in all media tested, the percentages of primordial follicles were reduced (P < 0.05) and the percentages of developing follicles were increased (P < 0.05) after 2 or 6 days of culture. Furthermore, culture of ovarian cortex for 6 days reduced the percentages of healthy, viable follicles when compared with the control (P < 0.05), except for cultures supplemented with IAA + EGF and EGF + FSH. In conclusion, the addition of IAA and EGF or EGF and FSH to the culture media were the most effective treatments to sustain the health and viability of activated ovine primordial follicles during in vitro culture. (c) 2005 Elsevier B.V. All rights reserved.

Effects of bovine Herpesvirus Type 5 on development of in vitro-produced bovine embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do alumínio no crescimento de brotações de Eucalyptus grandis x E. urophylla cultivadas in vitro

The aim of this work was to evaluate the effects of aluminum on the growth of Eucalyptus shoots cultivated in vitro through nutrient and total soluble protein content. The trial had a totally randomized design with four treatments and four replicates. The treatments were: 0.0; 0.25; 0.5 and 1.0 mM of AlCl 3.6H 2O. Shoots without roots of a Eucalyptus grandis x E urophylla clone were used for the in vitro culture. Evaluations were made on the 4th, 8th, 12th, 16th, 20th, 24th and 28th day of culture. The Al addition to the culture media reduced mainly Ca, P and K availability and absorptions by the shoots. The cellular metabolism was affected, conducted to morphological alterations in shoots (browning, mass calluses formation and shoots not friable), dry matter increased and a decreased in total protein soluble.