Effects of cryoprotectants and low temperatures on hatching and abnormal embryo development of Prochilodus lineatus (Characiformes: Prochilodontidae)

This study evaluated the effect of the cryoprotectants and the low temperatures on the embryonic development of Prochilodus lineatus, describing their main morphological alterations. On chilling sensitivity test, the survival rates at the twenty somites stage (20S) were 53.6% at 0ºC, and 100% in 5ºC. To test toxicity, the embryos were exposed to a graded series of 1,2-Propanediol (PROP), dimethyl sulfoxide (Me2SO4) and glycerol (GLY), terminating in a solution of high osmolarity. There was no significant difference in the embryos survival of toxicity test between series of PROP and Me2SO4 in the 6S and 20S. In the cooling protocols, were evaluated the effects of low temperature associated with cryoprotectants. At 5ºC, PROP showed survival rates above 75% in the gastrula stage (G) and above 90% in the 6S and 20S stages. High rates of abnormalities were observed, and the most recurrent were: small bodies, fins presenting uncontrolled cell growth, membrane rupture, and retraction. These results demonstrate the need to use cryoprotectant solutions, even when there is no ice nucleation, and, on the other hand, shows that high cryoprotectant concentrations promote numerous morphological lesions, compromising normal embryonic development.(AU)

Este estudo avaliou os efeitos dos crioprotetores e das baixas temperaturas no desenvolvimento embrionário de Prochilodus lineatus, descrevendo suas principais alterações morfológicas. Nos testes de sensibilidade ao frio, as taxas de sobrevivência, no estágio de vinte somitos (20S), foram de 53,6% a 0ºC e 100% a 5ºC. Nos testes de toxicidade, os embriões foram expostos a uma série graduada dos crioprotetores: 1,2-Propanediol (PROP), dimetilsulfóxido (Me2SO4) e glicerol (GLY), terminando em uma combinação de alta osmolaridade. Não houve diferença significativa na sobrevivência dos embriões nas séries do PROP e Me2SO4 nos estágios de 6S e 20S. Nos protocolos de resfriamento, foram avaliados os efeitos da baixa temperatura associados as séries crioprotetoras. A 5ºC, o PROP apresentou taxas de sobrevivência acima de 75% no estádio de gástrula (G) e acima de 90% nos estádios 6S e 20S. Foram observadas altas taxas de anormalidades sendo as mais recorrentes: corpos pequenos, nadadeiras apresentando crescimento celular descontrolado, ruptura da membrana e retração vitelínica. Estes resultados demonstram a necessidade do uso das soluções crioprotetoras, mesmo não havendo nucleação de gelo e, em contrapartida, demonstra que as elevadas concentrações crioprotetoras promovem numerosas lesões morfológicas, comprometendo o desenvolvimento embrionário.(AU)

Efeitos da idade da matriz leve e do período de armazenamento de ovos incubáveis no rendimento de incubação / Effects of ages of light breeder hens and storage period of hatchable eggs on the incubation efficiency

Avaliaram-se os efeitos da idade das matrizes leves e do período de armazenamento dos ovos sobre o rendimento de incubação. Foram utilizados 7.224 ovos da linhagem Dekalb, distribuídos de acordo com delineamento experimental inteiramente casualizado, sendo a altura de bandeja uma covariável. Os tratamentos foram definidos pelas idades das matrizes (32 e 57 semanas de idade) e o período de armazenamento dos ovos (três, cinco e sete dias), totalizando seis tratamentos com 14 repetições. A idade das aves influenciou o peso do ovo. Houve aumento na perda de peso dos ovos das matrizes de 32 e 57 semanas de idade, durante o armazenamento (10,48 e 14,16 por cento, respectivamente) e na transferência (10,40 e 12,07 por cento, respectivamente). Verificou-se maior taxa de eclosão (86,90 por cento) em ovos produzidos pelas matrizes novas. Independentemente da idade da matriz, à medida que aumentou o período de armazenamento, houve diminuição na taxa de eclosão dos ovos férteis (86,06 e 82,39 por cento, para as matrizes de 32 e 57 semanas de idade, respectivamente). As matrizes com 57 semanas de idade produziram pintos mais pesados e com menor relação peso do pinto/peso do ovo.

The effects of ages of light breeder hens and storage period of hatchabe eggs on the incubation efficiency were evaluated. A total of 7,224 Dekalb hatching eggs were assigned according to the sample random design, and the hatching tray height was considered a co-variable. The treatments were defined according to the breeder hens ages (32 and 57-week-old) and the period of storage of eggs (three, five, and seven days), with 14 replications. The age of the hen affected the weight of the eggs. There was loss of weight of the eggs of 32 and 57-week-old breeder hens during storage (10.48 and 14.16 percent, respectively) and transference (10.40 and 12.07 percent, respectively) according to the aging of the breeder hen. Eggs produced by the younger breeder hens had a higher hatching rate; however, no matter the age of the breeder hen, there was a decrease of the hatching rate of fertile eggs (86.06 and 82.39 percent, for the 32 and 57-week-old breeder hens, respectively) whenever the storage period was increased. The 57-weekold breeder hens produced heavier chicks and lower chick/egg weight coefficient.

Desempenho de novilhas Holandês na produção de embriões F1 Holandês-Zebu, em condições tropicais / Performance of Holstein heifers for F1 Holstein-Zebu embryo production, in tropical conditions

A produção in vivo de embriões bovinos é uma alternativa para a formação e manutenção de rebanhos mestiços F1 destinados à produção de leite. Objetivou-se estudar o potencial de utilização de novilhas da raça Holandês, mantidas em pasto e suplementadas, como doadoras de embrião, nas épocas de verão e inverno. Procederam-se superovulação e coleta de embriões em sete novilhas em cada época. Foram obtidos os dados climáticos, a temperatura retal e a frequência respiratória das doadoras. No verão, observou-se maior índice de temperatura e umidade (ITU) às 6h, 12h e 18h, bem como maior (P0,05) na média de estruturas recuperadas (20,14±17,47 vs. 8,57±9,40), embriões viáveis (14,57±11,91 vs. 7,14±7,24) e congeláveis (13,28±11,39 vs. 6,57±6,24) obtidos no inverno e no verão, respectivamente. Sob as condições estudadas, novilhas da raça Holandês podem ser utilizadas para a produção de embriões F1, tanto no verão, quanto no inverno.

In vivo embryo production (embryo transfer) is an alternative for F1 dairy herds formation and maintenance. The aim of this study was to evaluate the potential of utilization of grazing and supplemented Holstein heifers as embryo donors in summer or winter. Superovulation was performed in seven heifers on each season. Climatic parameters, as well as rectal temperature and respiratory rate of donors were measured. Temperature and humidity index (THI) was higher (p0.05). Holstein heifers can be used as embryo donors in both summer and winter, under the given conditions.

El embrión humano después de Dolly: Nuevas pautas para nuevos tiempos / No disponible

La posibilidad de clonar seres humanos trajo consigo múltiples problemas tanto para nuestros paradigmas éticos como para los sistemas jurídicos. En este artículo, nos centraremos en los cambios que nuestras construcciones normativas tendrán que implementar para adaptarse a la nueva situación planteada tras el nacimiento de Dolly. Defenderemos, en consecuencia, el cambio hacia una definición de embrión como el grupo de células capaces de desarrollarse hasta originar una persona como la mejor forma de adaptarse a los nuevos tiempos(AU)

The possibility of cloning human beings introduced a lot of issues in our ethical and legal frameworks. In this paper, we will put the focus into the necesary changes in the concept of embryo that our legal systems will have to implement in order to face the new situation. The description of the embryo as a group of cells able to develop pinto a human being will be defended here as the best way of doing so (AU)

Normal development of fetal hepatic haematopoiesis during the second trimester of gestation is upregulated by fibronectin expression in the stromal cells of the portal triads

Objetivo: en el segundo trimestre de la gestación, el principalfoco de hematopoyesis del feto es el hígado. En los órganoshematopoyéticos, las células del estroma, como fibroblastos,células epiteliales y células de tipo macrófago, desarrollan redespara mantener la hematopoyesis, es decir, la auto-renovación,la proliferación y el crecimiento de las células madre hematopoyéticas,al interactuar con las células progenitoras hematopoyéticas.Se sabe que las glucoproteínas de la MEC producidaspor las células del estroma desempeñan un papel crítico enla regulación del crecimiento y la diferenciación celulares. Sehan documentado numerosos factores solubles y de membranaque regulan directamente la hematopoyesis, pero se sabe pocode la actividad de las células del estroma hepático y de laproteína (fibronectina) de la matriz extracelular en el feto en relacióncon la hematopoyesis hepática. La unión de las célulaseritroides tardías a la fibronectina está bien tipificada y se creeque es crítica para las etapas terminales de la diferenciación eritroide.La intención de este artículo es determinar el papel dela fibronectina en la proliferación y diferenciación hematopoyéticadel hígado fetal en las distintas etapas del desarrollo.Material y método: examinamos y comparamos la expresióninmunohistoquímica de fibronectina en los campos portalesdel estroma hepático durante los trimestres primero, segundoy tercero del embarazo en relación con la aparición decélulas progenitoras hematopoyéticas CD34, progenitoras delestroma y endoteliales vasculares, respectivamente.Resultados: nuestros resultados mostraron una diferenciacuantitativa en cuanto a expresión de fibronectina en el estromadel tejido conjuntivo de los campos portales en el segundotrimestre de embarazo respecto al primero (p < 0,0001, pruebade la t) y respecto al tercero (p < 0,0001, prueba de la t). Sehallaron también cambios similares en cuanto a la expresión deCD34 respecto al primer (p < 0,0001, prueba de la t) y el tercertrimestres (p < 0,0001, prueba de la t), lo que indica la participacióndirecta de la fibronectina en el mantenimiento de laactividad hematopoyética

Objective: in midtrimester fetuses the principal site ofhematopoiesis is the liver. In hematopoietic organs, stromal cellssuch as fibroblasts, epithelial cells, and macrophage-like cells developnetworks to maintain hematopoiesis, i.e. hematopoietic stemcell self-renewal, proliferation, and growth, by interaction withhematopoietic progenitor cells. ECM glycoproteins produced by thestromal cells are known to play a critical role in the regulation of cellgrowth and differentiation. Numerous soluble and membraneboundfactors directly regulating haematopoiesis have been documented,but little is known about fetal hepatic stromal cell activityand stromal extracellular matrix protein-fibronectin, on fetal hepatichaematopoiesis. The binding of late stage erythroid cells to fibronectinhas been well characterized and is believed to be criticalfor the terminal stages of erythroid differentiation. The intention ofthis article is to determine the role of fibronectin in fetal hepatichematopoietic proliferation and differentiation in different stages ofdevelopment.Material and method: we examined and compared the immunohistochemicalexpression of fibronectin in the hepatic stromalportal fields in the 1st, 2nd, and 3rd trimester of gestation respectively,in relation to the appearance of CD34 progenitor hematopoietic,stromal progenitor and vascular endothelial positive cells.Results: our results demonstrated a quantitative difference inthe second trimester of gestation concerning the expression of fibronectinin the connective tissue stroma of the hepatic portalfields over the equivalent expression of the protein in the first (p <0.0001, t-test) and third trimester (p < 0.0001, t-test). Similarchanges in the above period were found concerning the expressionof CD34 during the second trimester of gestation, over thefirst (p < 0.0001, t-test) and third trimesters (p < 0.0001, t-test),suggesting a direct involvement of fibronectin in the sustaining ofhematopoietic activity

Semaphorin signaling facilitates cleft formation in the developing salivary gland.

Semaphorin signaling plays integral roles in multiple developmental processes. Branching morphogenesis is one such role that has not been thoroughly explored. Here, we show in mice that functional blockage of neuropilin 1 (Npn1) inhibits cleft formation in the developing submandibular gland (SMG) cultured ex vivo. This Npn1-dependent morphogenesis is mediated by Sema3A and Sema3C in an additive manner, and can be abolished by decreasing the expression of plexin A2 or plexin D1. VEGF, another known Npn1 ligand, has no apparent effects on SMG development. FGF signaling, which also mediates SMG branching morphogenesis, acts in parallel with semaphorin signaling. Finally, in contrast to the effect of FGF signaling, we find that semaphorins do not stimulate the proliferation of SMG epithelial cells. Instead, the semaphorin signals act locally on the epithelial cells to facilitate SMG cleft formation.

Retinoic acid is required for specification of the ventral eye field and for Rathke's pouch in the avian embryo.

We have investigated the role of retinoic acid (RA) in eye development using the vitamin A deficient quail model system, which overcomes problems of retinoic acid synthesising enzyme redundancy in the embryo. In the absence of retinoic acid, the ventral optic stalk and ventral retina are missing, whereas the dorsal optic stalk and dorsal retina develop appropriately. Other ocular abnormalities observed were a thinner retina and the lack of differentiation of the lens. In an attempt to explain this, we studied the expression of various dorsally and ventrally expressed genes such as Pax2, Pax6, Tbx6, Vax2, Raldh1 and Raldh3 and noted that they were unchanged in their expression patterns. In contrast, the RA catabolising enzymes Cyp26A1 and Cyp26B1 which are known to be RA-responsive were not expressed at all in the developing eye. At much earlier stages, the expression domain of Shh in the prechordal plate was reduced, as was Nkx2.1 and we suggest a model whereby the eye field is specified according to the concentration of SHH protein that is present. We also describe another organ, Rathke's pouch which fails to develop in the absence of retinoic acid. We attribute this to the down-regulation of Bmp2, Shh and Fgf8 which are known to be involved in the induction of this structure.

Morphology of the lateral pterygoid muscle associated to the mandibular condyle in the human prenatal stage

El músculo pterigoideo lateral (MPL) establece una relación directa con el cóndilo y el disco articular, desempeñando un rol importante en el movimiento mandibular a través del complejo articular temporomandibular (CATM). El objetivo de este trabajo fue determinar la correspondencia entre las características morfológicas de los haces musculares del MPL y el grado de mineralización del cóndilo mandibular en la etapa prenatal, mediante un análsisi estructgural, ultraestructural y microanalítico. Se utilizaron 16 fetos humanos de 11 a 37 semanas de gestación, procedentes de abortos espontáneos y sin patologías aparentes. Se obtuvieron muestras del músculo pterigoideo lateral y cóndilo mandibular, para ser examinadas meidante microscopía ñoptica, electrónica y técnicas de microanálisis. Muestras incluidas en parafina se emplearon en los estudios de inmunomarcación con desmina (dilución 1:25 Dako) y alfa actina sarcómerica (dilución 1:50 Dako), visualizados con sistema avidina-biotina. El lado contralateral de cada una se utilizó para microscopía electrónica de transmisión. En 4 cóndilos de 17 y 21 semanas de desarrollo se analizó el contenido relativo de calcio y fósforo con la técnica microanalítica por difracción de rayos X. Entre las 11 y 16 semanas, el MPL estuvo contituido por miotubos secundarios asociados a céluals satélites y fascículos. La organización del músculo a las 37 semanas fue más compleja, con aspecto multipeniforme y con la edad se redujo la intensidad de la inmunomarcación con alfa actina sarcomérica. Estos resultados sugieren que entre las 16 y 22 semanas, el proceso de diferenciación y maduración de las fibras musculares precede y prevalece sobre el desarrollo y mineralización del cóndilo mandibular. La funcionalidad aún rudimentaria del MPL prenatal sería uno de los factores reguladores del proceso de osificación a nivel del cóndilo mandibular, cuyo ritmo aumentaría a partir de las 22 semanas de gestación (AU)

Morphology of the lateral pterygoid muscle associated to the mandibular condyle in the human prenatal stage

El músculo pterigoideo lateral (MPL) establece una relación directa con el cóndilo y el disco articular, desempeñando un rol importante en el movimiento mandibular a través del complejo articular temporomandibular (CATM). El objetivo de este trabajo fue determinar la correspondencia entre las características morfológicas de los haces musculares del MPL y el grado de mineralización del cóndilo mandibular en la etapa prenatal, mediante un análsisi estructgural, ultraestructural y microanalítico. Se utilizaron 16 fetos humanos de 11 a 37 semanas de gestación, procedentes de abortos espontáneos y sin patologías aparentes. Se obtuvieron muestras del músculo pterigoideo lateral y cóndilo mandibular, para ser examinadas meidante microscopía ñoptica, electrónica y técnicas de microanálisis. Muestras incluidas en parafina se emplearon en los estudios de inmunomarcación con desmina (dilución 1:25 Dako) y alfa actina sarcómerica (dilución 1:50 Dako), visualizados con sistema avidina-biotina. El lado contralateral de cada una se utilizó para microscopía electrónica de transmisión. En 4 cóndilos de 17 y 21 semanas de desarrollo se analizó el contenido relativo de calcio y fósforo con la técnica microanalítica por difracción de rayos X. Entre las 11 y 16 semanas, el MPL estuvo contituido por miotubos secundarios asociados a céluals satélites y fascículos. La organización del músculo a las 37 semanas fue más compleja, con aspecto multipeniforme y con la edad se redujo la intensidad de la inmunomarcación con alfa actina sarcomérica. Estos resultados sugieren que entre las 16 y 22 semanas, el proceso de diferenciación y maduración de las fibras musculares precede y prevalece sobre el desarrollo y mineralización del cóndilo mandibular. La funcionalidad aún rudimentaria del MPL prenatal sería uno de los factores reguladores del proceso de osificación a nivel del cóndilo mandibular, cuyo ritmo aumentaría a partir de las 22 semanas de gestación (AU)

Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis.

Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.

Ontogeny of the nasopalatine duct in primates.

Ecological explanations have been put forward to account for the precocious or delayed development of patency in ducts leading to the vomeronasal organ (VNO) in certain mammals. Perinatal function may be related, in part, to the patency or fusion of the vomeronasal and nasopalatine (NPD) ducts. However, few studies have focused on NPD development in primates, which generally have a prolonged period of dependence during infancy. In this study we examined 24 prenatal primates and 13 neonatal primates, and a comparative sample of fetal mice and insectivores. In embryonic and early fetal Microcebus murinus, the NPD was completely fused, whereas in fetuses of later stages the duct was partially fused or completely patent. M. myoxinus of all stages demonstrated some degree of NPD fusion. In all other prenatal primates, the NPD was fused to some extent. Four prenatal insectivores (Tenrec ecaudatus) showed some degree of NPD fusion. In Mus musculus at 19 days gestation, the NPD was patent, although the anatomically separate VNO duct was fused. T. ecaudatus and most of the neonatal primates revealed complete NPD patency. An exception was Saguinus geoffroyi, which exhibited fusion of the NPD near the VNO opening. These observations may relate to differences in perinatal VNO function. The differences noted in our study suggest that M. murinus and M. myoxinus may differ in perinatal VNO functionality and perhaps in related behavior. Observations of neonatal primates suggest that NPD patency may be relatively common at birth and could serve other purposes in addition to being an access route for VNO stimuli.

Two Pax genes, eye gone and eyeless, act cooperatively in promoting Drosophila eye development.

We report the identification of a Drosophila Pax gene, eye gone (eyg), which is required for eye development. Loss-of-function eyg mutations cause reduction or absence of the eye. Similar to the Pax6 eyeless (ey) gene, ectopic expression of eyg induces extra eye formation, but at sites different from those induced by ey. Several lines of evidence suggest that eyg and ey act cooperatively: (1) eyg expression is not regulated by ey, nor does it regulate ey expression, (2) eyg-induced ectopic morphogenetic furrow formation does not require ey, nor does ey-induced ectopic eye production require eyg, (3) eyg and ey can partially substitute for the function of the other, and (4) coexpression of eyg and ey has a synergistic enhancement of ectopic eye formation. Our results also show that eyg has two major functions: to promote cell proliferation in the eye disc and to promote eye development through suppression of wg transcription.

Perturbation of extracellular matrix prevents association of the otic primordium with the posterior rhombencephalon and inhibits subsequent invagination.

In the avian embryo, the otic primordia become visible by Hamburger and Hamilton stage 10 as a pair of thickened regions of head ectoderm. In contrast to other epithelial primordia, invagination occurs by means of formation of a series of folds in distinct areas of the primordium, giving the otic vesicle a box-like appearance. Because previous work has shown that otic invagination is ATP and calcium independent, it is unlikely that cytoskeletal changes are the primary mechanism responsible for invagination as in other epithelial primordia. Interaction of the primordium with surrounding tissues may provide the force for otic invagination. These extracellular forces may be transduced through extracellular matrix macromolecules and their cell surface receptors. This investigation tests the hypothesis that fusion of the otic and hindbrain basal laminae between stages 11 and 13 is necessary for normal invagination. Perturbation of binding of the otic primordium to the neural tube was accomplished by means of microinjection of antibodies to various extracellular matrix components and integrin subunits into the head mesenchyme in the otic region at stage 10. Only antibodies to laminin and integrins caused detachment of the otic primordium from the hindbrain. These experiments suggest that fusion of the otic and hindbrain basal laminae is required for subsequent invagination and, furthermore, that this event is mediated by components of the extracellular matrix.

El desarrollo embrionario preimplantatorio tras FIV es superior a icsi debido a la calidad espermática y no a la técnica / Superior preimplantation development of embryos after in vitro fertilization (IVF) compared to intracytoplasmic sperm injection (ICSI) is not due to tbe tecbnique but to the sperm quality

Objetivo: Determinar el impacto de la técnica de ICSI sobre el desarrollo embrionario, estudiando el desarrollo preimplantatorio hasta el estadio de blastocisto, comparando embriones obtenidos mediante FIV convencional con los obtenidos mediante ICSI con muestras de semen con alteraciones severas o aparentemente normales. Métodos: Se estudió el desarrollo preimplantatorio de un total de 900 embriones humanos de día 2 que se sometieron a co-cultivo y transferencia en estadio de blastocisto. Primero, se analizaron 852 embriones derivados de ovocitos donados de mujeres jóvenes y fértiles en los cuales la técnica de inseminación artificial (FIV o ICSI) se decidió en base a las características del semen del marido de la receptora. Se dividieron los embriones en los obtenidos mediante FIV convencional (n = 311), y embriones obtenidos mediante ICSI (n = 541). Segundo, se compararon con un total de 48 embriones de día dos obtenidos de ovocitos hermanos y muestras de esperma normales. Resultados: El porcentaje de formación de blastocistos fue significativamente mayor en los embriones obtenidos mediante FIV convencional (67,1 por 100) comparado con aquellos obtenidos mediante ICSI (59,0 por 100) (p < 0,05). La tasa de embarazo, implantación y aborto fueron similares en las receptoras de ambos grupos (ya que se transfirió el mismo número de blastocistos). Además, evaluamos la tasa de formación de blastocistos obtenidos por FIV (45,2 por 100) e ICSI (53,3 por 100), provenientes de ovocitos hermanos y muestras de esperma normales, y no encontramos diferencia estadísticamente significativa.Conclusiones: Este estudio indica que el desarrollo preimplantatorio, demostrado por el porcentaje de formación de blastocistos es superior en los embriones obtenidos por FIV que por ICSI. Además, este estudio sugiere que este efecto es debido a la calidad del esperma, y no a las condiciones técnicas del procedimiento de ICSI (AU)

Produçäo de embriöes a partir do desenvolvimento de oócitos imaturos in vitro: valor de estimulaçäo curta com FSH / In vitro development of immature oocytes to viable embryos: brief ovarian stimulation with FSH

OBJETIVO: Avaliar os recentes avanços nas técnicas de maturaçäo oocitária que têm possibilitado o desenvolvimento de embriöes a partir de oócitos imaturos. Desta forma objetivamos avaliar a maturaçäo ovular, taxas de fertilizaçäo e desenvolvimento embrionário em oócitos imaturos aspirados sob pequenas doses de estímulo gonadal. MATERIAL E MÉTODOS: 8 mulheres entre 28 e 39 anos foram submetidas a estimulaçäo com urofolitrofina purificada (Metrodin) e seus oócitos coletados quando pelos menos 2 deles atingiram diâmetro médio de 10mm. Os oócitos coletados foram cultivados em meio TCM-199 suplementado com soro sintético. Metrodin, Profasi (hCG) e Piruvato, ambiente controlado a 37 graus Celsius e concentraçäo de CO2 a 5 por cento por 24 até 72h. Após avaliaçäo da maturidade ovular, procedeu-se a ICSI e transferência de embriöes morfologicamente normais por via transcervical para cavidade uterina. RESULTADOS: 43 oócitos coletados de 7 pacientes foram submetidos a maturaçäo in vitro. Em uma das pacientes näo evidenciou presença de oócitos. Observou-se desenvolvimento até primeiro corpúsculo polar em 27 (63 por cento) ocócitos, sendo que destes, 17 (63 por cento) fertilizaram e 15 (88 por cento) apresentaram clivagem em seu seguimento. Como resultado, obtivemos 1 gravidez após 6 transferências (1/6 - 17 por cento por transferência) que resultaram em gestaçäo viável, com evoluçäo normal, atualmente no segundo trimestre. CONCLUSÖES: Estes resultados demonstram a viabilidade de cultura e conseqüente maturaçäo in vitro de oócitos imaturos, observando-se resultados em termos de taxas de fertilizaçäo, clivagem e gravidez, próximas as encontradas em ciclos induzidos até o completo desenvolvimento ovular.

Effect of embryo density and growth factors on in vitro preimplantation development of mouse embryos

Embryo development depends on maternal and embryonic factors that may regulate genetic programs in early development. Effects of growth factors on proliferation, differentiation and morphogenesis along embryogenesis have been documented. However, studies have not established the role of growth factors in the preimplantational period. The purpose of this study was to investigate the possible effects of growth factors and embryo density on mouse preimplantation development in vitro. Two-and eight-cell CF-1 embryos were cultured individually or in groups of ten in HTF medium, alone or with EGF, TGF-beta1 and IGF-I. Cleavage rate varied greatly with growth factors and increased significantly when eight-cell embryos were cultured in groups. On the other hand, when two-cell embryos were cultured in group, the cleavage rate was slower than that obtained when embryos were individually cultured. The differentiation rate increased significantly in two-cell embryos cultured in groups (p<0.05). EGF, TGF-beta1 and IGF-I increased differentiation rates significantly in two-cell embryos individually cultured for 68 hours. The combination of EGF and TGF-beta1 increased the differentiation rates significantly. The other combinations were not effective in modifying this parameter. Hatching rates increased in embryos cultured in groups (p<0.05). TGF-beta1 decreased this parameter significantly in two-or eight-cell embryos cultured in groups (p<0.05). The data described in this report suggest that preimplantational mouse embryos produce some factor or factors that enhance its development, specially the differentiation and hatching rates. However, a functional role for polypeptide growth factors during preimplantational development has to be determined.

Desenvolvimento, in vitro, de blastômeros isolados e embriöes humanos, pós-biopsia, no período pré-implantaçäo / Development, in vitro, of isolated blastomeres and human embryos, pos biopsy, in preimplantation period

Embriöes humanos normalmente fertilizados foram biopsiados, em estágio de clivagem, no dia 3 após fertilizaçäo in vitro (estágio de 6 a 10 células). Vinte e oito embriöes tiveram 1 blastômero removido e 19 embriões, 2 blastômeros, que foram colocados em co-cultura com os embriöes biopsiados. Embriöes e blastômeros foram analisados diariamente para avaliaçäo do desenvolvimento morfológico até o dia 6, quando o número de células foi contado, através de marcaçäo nuclear. No total, 53 por cento dos embriões biopsiados (25/47), atingiram o estágio de blastocisto entre o dia 5 e o dia 6, e a proporçäo foi a mesma, independente do número de células removidas. O número total de células no estágio de blastocisto (56,2 ñ 3,0; 64,7 ñ 5,5) e o número de células do trofectoderma (45,4 ñ 3,5; 44,0 ñ 5,7) e do embrioblasto (14,0 ñ 1,2; 16,6 ñ 1,8) näo foram significantemente diferente nos embriões que tiveram 1 ou 2 células removidas, respectivamente. Setenta e dois por cento dos blastômeros dividiram-se pelo menos uma vez, no período de 3 dias em cultura, e 50 por cento dividiram-se mais de uma vez. O número total de células após 3 dias em cultura foi de 4,8 ñ 0,51 por blastômero quando uma célula foi removida, e 8,3 ñ 1,0 quando duas células foram removidas. Cinquenta e cinco por cento dos blastômeros cavitaram neste período. Dos blastômeros removidos de embriöes que se desenvolveram até o estágio de blastocisto, 92 por cento sofreram divisäo e 76 por cento cavitaçäo. Naqueles removidos de embriöes que näo se desenvolveram até o estágio de blastocisto, 50 por cento sofreram divisäo (p<0,002) e 32 por cento cavitaçäo (p<0,003). Oito por cento dos blastômeros do primeiro grupo e 41 por cento dos blastômeros do segundo, näo sofreram divisäo ou cavitaçäo (p<0,008). Concluindo, a remoçäo de 1 ou 2 blastômeros, pela técnica de biopsia de embriöes humanos em estágio de 8 células, näo afeta adversamente o desenvolvimento embrionário, sendo que näo existe diferença em se remover 1 ou 2 células; blastômeros isolados, biopsiados de embriöes humanos em estágio de 8 células, possuem a capacidade de proliferar, formar cavidade e diferenciar em células do trofectoderma, in vitro, quando mantidos em co-cultura com os embriöes de origem e; blastômeros de um mesmo embriäo possuem um desenvolvimento sincrônico e equivalente, mesmo quando isolados e cultivados externamente ao embriäo de origem...

Effect of embryo density and growth factors on in vitro preimplantation development of mouse embryos

Embryo development depends on maternal and embryonic factors that may regulate genetic programs in early development. Effects of growth factors on proliferation, differentiation and morphogenesis along embryogenesis have been documented. However, studies have not established the role of growth factors in the preimplantational period. The purpose of this study was to investigate the possible effects of growth factors and embryo density on mouse preimplantation development in vitro. Two-and eight-cell CF-1 embryos were cultured individually or in groups of ten in HTF medium, alone or with EGF, TGF-beta1 and IGF-I. Cleavage rate varied greatly with growth factors and increased significantly when eight-cell embryos were cultured in groups. On the other hand, when two-cell embryos were cultured in group, the cleavage rate was slower than that obtained when embryos were individually cultured. The differentiation rate increased significantly in two-cell embryos cultured in groups (p<0.05). EGF, TGF-beta1 and IGF-I increased differentiation rates significantly in two-cell embryos individually cultured for 68 hours. The combination of EGF and TGF-beta1 increased the differentiation rates significantly. The other combinations were not effective in modifying this parameter. Hatching rates increased in embryos cultured in groups (p<0.05). TGF-beta1 decreased this parameter significantly in two-or eight-cell embryos cultured in groups (p<0.05). The data described in this report suggest that preimplantational mouse embryos produce some factor or factors that enhance its development, specially the differentiation and hatching rates. However, a functional role for polypeptide growth factors during preimplantational development has to be determined. (AU)