Felipe Ledur Ongaratto

Survival rates of mouse blastocyst vitrified in dimethylformamide based solutions associated with ethylene glicol or 1-2 propanediol

O objetivo deste estudo foi determinar o efeito da dimetilformamida (DF) associada com etileno glycol (EG) ou 1-2 propanediol (PROH) durante a vitrificacao, no desenvolvimento in vitro de blastocistos murinos. A toxicidade dos crioprotetores foi avaliada ao expor os embrioes as tres solucoes de equilibrio (ES) compostas pelas misturas de DF, EG ou PROH (10% v/v de cada) em mPBS + 0,5% PVA, em diferentes intervalos de tempo (1, 3 e 10min). Em um segundo experimento, os embrioes foram expostos as mesmas ES (durante 1 e 3min), seguido da exposicao as tres respectivas solucoes de vitrificacao (VS) (20% v/v de cada) durante 30seg. Apos 72 horas de cultivo in vitro, as taxas de expansao e eclosao dos embrioes expostos durante os periodos de 1 e 3min as solucoes de equilibrio ES1 e ES2 foram semelhantes. No entanto, os embrioes expostos durante 10min as solucoes de equilibrio com DF apresentaram taxas de sobrevivencia inferiores a solucao de EG-PROH (P<0,01). Alem disso, as taxas de sobrevivencia dos embrioes expostos a DF-PROH (ES+VS) foram menores que as dos embrioes expostos as outras solucoes (P<0,01). A vitrificacao dos blastocistos foi realizada apos a exposicao dos embrioes nas tres ES+VS (por 1min e 30seg, respectivamente), usando micropipetas de vidro (GMP). As taxas de sobrevivencia foram menores nos blastocistos vitrificados nas solucoes compostas por DF (3%-3/108 e 17,1%-19/111), em relacao a solucao EG-PROH (69%-73/105) (P<0,01). Em conclusao, a DF adicionada como crioprotetor as solucoes de vitrificacao apresenta efeitos deleterios na capacidade de desenvolvimento in vitro dos blastocistos murinos vitrificados.

Probability, odds and random chance: the difficult task of modulating the epigenetic profile of cloned embryos

Since the beginning of modern embryology, scientists have wondered about how a small number of totipotent embryonic cells can become an individual with a wide variety of organs and tissues with distinct functions. Also, the idea of generating a cloned animal using a nucleus from a donor cell is not recent. However, it has taken years of research to achieve this goal, especially regarding mechanisms of cell reprogramming required to return a differentiated cell to totipotency. Cloning by somatic cell nuclear transfer (SCNT) has been a valuable tool to understand epigenetic mechanisms related to cellular reprogramming. However, cloning efficiency is still low, with a low percentage of embryos resulting in healthy animals. The high attrition rate is associated with incomplete or abnormal epigenetic reprogramming, such that many cloned embryos have DNA methylation patterns different than controls, resulting in faulty gene expression and subsequent developmental failures. Attempts to improve genome reprogramming by modulation of oocyte quality and/or somatic cell plasticity, thereby increasing cloning efficiency and preventing detrimental effects on development, have proven ineffective. The recent development of DNA editing techniques may facilitate an improved understanding of cellular reprogramming and the role of DNA methylation in development. These novel tools may lead to new means to modulate epigenetic programming and inheritance, and hold great promise to assist in epigenetic remodeling of the donor nucleus. Such strategies are likely to improve the odds for successful cloning.

Vitrification of immature and matured bovine oocytes: effect of brilliant cresyl blue selection and hyaluronan addition

The aim of the present study was to evaluate the effect of brilliant cresyl blue (BCB) selection, the type of oocyte (immature or matured) and the use of hyaluronan in the vitrification solution on further embryo developmental competence. Oocytes (n = 1308) obtained from abattoir ovaries were classified by BCB stain. Control oocytes were maintained in holding media for 90 min and then subdivided to be placed into maturation media without any treatment or were vitrified. Immature or matured oocytes were vitrified by the solid-surface technique using two different vitrification solutions. VS1: composed of 10% ethylene glycol (EG) for 10 min followed by 20% EG + 0.2M trehalose for 30 sec and finally into 30% EG + 0.5M trehalose for 30 sec, or VS2 composed by 10% EG for 10 min, followed by 20% EG + 0.2M trehalose for 30 sec, and finally into 30% EG+ 0.5M trehalose + 0.1 g/ml hyaluronan for 30 sec. Oocytes were then loaded into Fyberplugs™ and vitrified. After one week, Fyberplugs™ were open and placed directly into (37°C) 0.5M sucrose solution for 5 min, then into 0.25M of sucrose for another 5 min and finally placed into maturation medium for in vitro production. Cleavage and development rates were examined on days 2 and 7 after fertilization, respectively. The blastocyst rate of vitrified oocytes selected as BCB + (5.5 ± 0.6%) were higher than those selected as BCB - (1.0 ± 0.4%) and those that were not selected by BCB (2.0 ± 1.1%; P < 0.001). Furthermore, immature vitrified oocytes had greater (P < 0.05) cleavage and blastocyst rates (44.8 ± 1.9% and 4.0 ± 0.6%) than matured vitrified oocytes (38.3 ± 2.8% and 2.5 ± 0.6%). Finally, the addition of hyaluronan to the vitrification solution had no significant effect on development rates. In conclusion, the selection of oocytes by BCB and the use of immature oocytes increase the development rates of vitrified-warmed oocytes.