Name: collection and cryopreservation of hamster oocytes and mouse embryos
Uploaded: 2009-03-27T00:00:00.000+00:00
Duration: 13 min 36 s
Description: Watch this Scientific Journal Video about Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos at JoVE.com

Este trabalho foi financiado pelo Ministerio de Educación y espanhol Ciencia (BIO 2006-11792) e da Generalitat de Catalunya (2005-SGR00437). Autores gostariam de agradecer Marc Puigcerver e Jonatan Lucas para a sua assistência técnica na preparação dos meios de comunicação. Todo o pessoal da d&#39;Servei Estabulari é reconhecido por alojamento dos animais e, especialmente, Juan Jose Martin e Javier Benito para a sua contribuição adicional sobre a gravação parte deste vídeo. BCN é um companheiro da Fundação Português parágrafo a Ciência e Tecnologia e SG é um companheiro do espanhol Ministerio de Educación y Ciencia.

Cuidados com os animais e os procedimentos foram realizados de acordo com as diretrizes e as normas aprovadas pelo Comitê de Ética em Pesquisa Animal e Humana da Universitat Autònoma de Barcelona, ​​Espanha. I. coleta de oócitos <ol><li> Verifique e selecione hamsters feminino (Mesocricetus auratus; 12/08 semana de idade) da cepa de Ouro da Síria para a presença de um corrimento vaginal espesso (descarga pós-ovulatória). </li><li> Induzir fêmeas selecionadas para superovulate por injeção intraperitoneal de 40 UI de PMSG (Pregnant Mare Serum Gonadotropin), seguido de 40 UI de hCG (gonadotropina coriônica humana) 60 horas depois. </li><li> Euthanize hamsters fêmeas 16 horas após a administração de hCG em uma câmara de dióxido de carbono. </li><li> Isolar ovidutos das fêmeas, como mostrado no vídeo e transferi-los para uma queda de KSOM-H médio. </li><li> Coletar oócitos cumulus complexos rasgando a ampola do oviduto sob um microscópio estereoscópico em uma gota de hialuronidase (156 U / ml) a 37 ° C. Oócitos serão liberados a partir de células cumulus em cerca de 5 minutos. </li><li> Pick up ovócitos de hialuronidase e lavá-los duas vezes em KSOM-H médio. </li></ol> Para a coleta de embriões de camundongos, ratos fêmeas (Mus musculus; 6-8 semana de idade) são induzidos a superovulate por injeção intraperitoneal de 5 UI de PMSG seguido por 5 UI de hCG 48 horas mais tarde e acasaladas com ratos machos. As fêmeas são sacrificadas por deslocamento cervical em 24-26, 44-46 ou 54-56 horas após a administração de hCG para obtenção de pronuclear, 2 de células ou embriões de 4 células estágio, respectivamente. Colheita de embriões pronuclear é realizado exatamente como descrito para oócitos de hamster. Embriões nas fases 2 e 4 células de células são recolhidas através de lavagem com o ovidutos KSOM-H médio [6]. Nota: Na sequência deste protocolo aproximadamente 20 a 30 oócitos / embriões podem ser coletadas de cada fêmea. II. Preparação de soluções de congelamento e descongelamento <ol><li> Prepare solução que eu diluindo 0,57 ml de propilenglycol em 5 ml de KSOM-H (1,5 M de propilenglycol). </li><li> Prepare a solução de descongelamento, adicionando 2 ml de KSOM-H para um tubo com 0,2052 g de sacarose (0,3 M de sacarose). </li><li> Preparar a solução de congelamento, adicionando 2 ml de solução I a um tubo com 0,0684 g de sacarose (1,5 M de sacarose e 0,1 propilenglycol M). </li></ol> Nota: Eu Solution e congelamento e descongelamento, as soluções devem ser preparadas imediatamente antes de seu uso. No entanto, as quantidades especificadas de sacarose a ser usada para a preparação do congelamento e descongelamento soluções podem ser ponderados com antecedência e armazenado em 3 ml de tubos em temperatura ambiente por vários meses. III. Protocolo de congelamento <ol><li> Ovócitos transferência / embriões a partir do meio KSOM-H para uma gota de solução I e incubar por 7-15 minutos em temperatura ambiente. </li><li> Enquanto isso, prepare uma placa de Petri 90 milímetros para carregar a palha, colocando 90 gotas mL de congelamento e descongelamento soluções como é mostrado no vídeo (1 gota de cada solução / palha). </li><li> Transferir o número desejado de oócitos / embriões para as gotas de solução de congelamento. Nota: Nós normalmente transferência entre 5 a 30 oócitos ou embriões / gota. </li><li> Anexar uma palha para um sistema de aspiração de boca controlada e carregá-lo seguindo esta ordem: solução I, do ar, queda de congelamento com os oócitos, ar, queda de descongelamento, o ar. </li><li> Uma vez que a palha é carregada, manter aspiração até que a primeira gota introduzido (solução I) atinge o polímero no topo do canudo e sela o fim. Termo-selar o outro lado da palha ou selar com um plugue de plástico. </li><li> Ligue o freezer programável e selecione o programa de congelamento. O programa de congelamento utilizado neste protocolo é baseado nas taxas de resfriamento publicado originalmente por Lassalle et al. [7]: <ul><li> 2 ° C / min da temperatura ambiente até -7 ° C 0 ° C / min para 10 min para permitir equilíbrio de temperatura e indução de semeadura (veja abaixo) </li><li> 0,3 ° C / min de -7 ° C a -30 ° C 0 ° C / min por 2 min para permitir equilíbrio de temperatura </li><li> 35 ° C / min de -30 ° C a -130 ° C </li></ul></li><li> Carregar os canudos nos slots do titular do congelador programável, certificando-se que a parte da palha com a gota de solução descongelamento é voltado para fora. Inicie o programa de congelamento. </li><li> Quando a temperatura da amostra atinge -7 ° C ea refrigeração está em espera, executar semeadura manual: mergulhar a pinça em nitrogênio líquido, puxe os titulares um pouco fora do freezer para expor a parte da palha contendo as gotas de solução de descongelamento, e imediatamente toque nesta parte do canudos com o fórceps. Repita a operação para cada titular e cada um de palha no suporte, e deixar que o programa de congelamento de correr até o fim. </li><li> Quando o programa estiver concluído, mergulhe os titulares em nitrogênio líquido. </li><li> Retire as palhas datitulares e transferi-los para um gobelet rotulados. </li><li> Finalmente, guarde o gobelet dentro de um tanque de nitrogênio líquido. </li></ol> IV. Descongelamento protocolo <ol><li> Remover a palha do tanque de nitrogênio líquido. </li><li> Manter a palha em temperatura ambiente por 40 segundos. </li><li> Mergulhe a palha em banho-maria a 30 ° C durante 40 segundos. </li><li> Retire a ficha da palha ou cortar a ponta termo-selado. </li><li> Esvaziar a palha numa placa de Petri de 35 mm e misture delicadamente as duas gotas que contêm o congelamento e descongelamento soluções. </li><li> Deixe oócitos / embriões na mistura por 15 minutos em temperatura ambiente. </li><li> Transferência de oócitos / embriões frescos para uma queda de KSOM-H para um adicional de 15 minutos a 37 ° C. </li><li> Oócitos / embriões estão agora prontos para manipulação posterior. </li></ol> Nota: Durante as etapas 6 e 7 você verá que os oócitos descongelados / embriões lentamente inchar devido à reidratação. Representante Resultados V. O uso do protocolo de criopreservação aqui relatados resulta em uma taxa de sobrevivência de cerca de 95% para oócitos de hamster e de&gt; 90%, 85% e 80% para mouse B6CBAF1 embriões no pronuclear, 2-estágios e de célula de 4 células, respectivamente. Quando embriões de camundongos são cultivadas in vitro em KSOM após o descongelamento, pelo menos 70 a 80% devem chegar ao estágio de blastocisto em condições de cultura ideal. É recomendado que você incluir um grupo controle (palha) de oócitos / embriões em cada experimento de congelamento e descongelamento que este grupo antes do resto dos oócitos / embriões são usados ​​para manipulação posterior. Se as taxas de sobrevivência para o grupo controle é menor do que o esperado, descongele outra palha do mesmo lote. Se as taxas de sobrevivência para este segundo grupo também são baixos, descarte as palhetas restantes deste lote de congelamento.

<ol>
	<li>Whittingham, D. G., Leibo, S. P., Mazur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+mouse+embryos+frozen+to+-196+and+-269+&#176;C.">Survival of mouse embryos frozen to -196 and -269 &#176;C.</a> Science. 178, 411-414 (1972).</li><li>Wilmut, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cooling+rate,+warming+rate,+cryoprotective+agent+and+stage+of+development+on+survival+of+mouse+embryos+during+freezing+and+thawing.">The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing.</a> Life Sci. 11, 1071-1079 (1972).</li><li>Parkening, T. A., Tsunoda, Y., Chang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+various+low+temperatures,+cryoprotective+agents+and+cooling+rates+on+the+survival,+fertilizability+and+development+of+frozen-thawed+mouse+eggs.">Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse eggs.</a> J. Exp. Zool. 197, 369-374 (1976).</li><li>Schneider, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryobiological+principles+of+embryo+freezing.">Cryobiological principles of embryo freezing.</a> J. In Vitro Fert. Embryo Transf. 3, 3-9 (1986).</li><li>Vajta, G., Kuwayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+cryopreservation+systems.">Improving cryopreservation systems.</a> Theriogenology. 65, 236-244 (2006).</li><li>Duselis, A. R., Vrana, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrieval+of+mouse+oocytes.">Retrieval of mouse oocytes.</a> JoVE. 3, (2007).</li><li>Lassalle, B., Testart, J., Renard, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryo+features+that+influence+the+success+of+cryopreservation+with+the+use+of+1,2+propanediol.">Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol.</a> Fertil. Steril. 44, 645-651 (1985).</li><li>Ib&#225;&#241;ez, E., Grossmann, M., Vidal, F., Egozcue, J., Santal&#243;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+caprine+beta-lactoglobulin+transgenic+mouse+embryos.">Cryopreservation of caprine beta-lactoglobulin transgenic mouse embryos.</a> Cryobiology. 35, 290-298 (1997).</li><li>Dinnyes, A., Wallace, G. A., Rall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genotype+on+the+efficiency+of+mouse+embryo+cryopreservation+by+vitrification+or+slow+freezing.">Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing.</a> Mol. Reprod. Dev. 40, 429-435 (1995).</li><li>Tateno, H., Kamiguchi, Y., Mikamo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+freezing+and+thawing+method+of+hamster+oocytes+designed+for+both+the+penetration+test+and+chromosome+assay+of+human+spermatozoa.">A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa.</a> Mol. Reprod. Dev. 33, 202-209 (1992).</li><li>Biggers, J., McGuinnis, L. K., Raffin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+and+preimplantation+development+of+the+mouse+in+the+protein-free+KSOM.">Amino acids and preimplantation development of the mouse in the protein-free KSOM.</a> Biol. Reprod. 63, 281-293 (2000).</li></ol>

Com este protocolo de oócitos e embriões hamster rato pode ser criopreservados com sucesso. Uma vez congelados, os oócitos / embriões podem ser armazenados em tanques de nitrogênio líquido por tempo indeterminado e recuperado a qualquer momento ou local desejado. Isto oferece a vantagem de ter amostras quase pronto para uso sempre que necessário. Por outro lado, este protocolo também pode ser usado para gerar cryobanks embrião de linhagens de camundongos valiosos (como linhagens transgênicas), como uma alternativa econômica e seg...

<table><tbody><tr><td>Material Name</td><td>Type</td><td>Company</td><td>Catalogue Number</td><td>Comment</td></tr><tr><td>French type mini-straw 0.25 ml</td><td></td><td>Mini-tub</td><td>13407/0010</td><td></td></tr><tr><td>hCG</td><td></td><td>Farma-Lepori, Spain</td><td>749036.4</td><td></td></tr><tr><td>Hyaluronidase</td><td></td><td>Sigma</td><td>H-3884</td><td></td></tr><tr><td>KSOM-H medium</td><td></td><td></td><td></td><td>Ref. [11]</td></tr><tr><td>Liquid Nitrogen </td><td></td><td>Air Liquide</td><td></td><td></td></tr><tr><td>Plastic plugs</td><td></td><td>Mini-tub</td><td>19046</td><td></td></tr><tr><td>PMSG</td><td></td><td>Intervet, Spain</td><td>1.614/8447</td><td></td></tr><tr><td>Propilenglycol</td><td></td><td>Fluka</td><td>82280</td><td></td></tr><tr><td>Sucrose</td><td></td><td>Merck</td><td>107687</td><td></td></tr><tr><td>Surgical Scissors</td><td></td><td>Aesculap</td><td>BC-060R; BG-061R</td><td></td></tr><tr><td>Thermo-sealer</td><td></td><td>SIZ</td><td>220</td><td></td></tr><tr><td>35 and 90 mm culture dishes</td><td></td><td>Nunc</td><td>153066; 150350</td><td></td></tr><tr><td>10 ml tubes</td><td></td><td>Greiner Bio-one</td><td>163160</td><td></td></tr><tr><td>Nitrogen tank</td><td></td><td>MVE, Cryogenics</td><td>XC 43/28</td><td></td></tr><tr><td>Programmable Freezer</td><td></td><td>Planner Products Ltd.</td><td>Kryo-10 Series III</td><td></td></tr><tr><td>Forceps</td><td></td><td>Aesculap</td><td>BD-331R; BD-053R; OC-020R</td><td></td></tr></tbody></table>

collection and cryopreservation of hamster oocytes and mouse embryos

Embriões e oócitos criopreservados com sucesso pela primeira vez mais de 30 anos atrás, quando Whittingham<i&gt; Et al.</i&gt;<sup&gt; 1</sup&gt; E Wilmut<sup&gt; 2</sup&gt; Separadamente descrito que embriões de camundongos poderiam ser congeladas e armazenadas a -196 ° C e, alguns anos mais tarde, Parkening<i&gt; Et al</i&gt;.<sup&gt; 3</sup&gt; Relatou o nascimento de filhotes vivos resultantes de fertilização in vitro (FIV) de oócitos criopreservados. Desde então, o uso de técnicas de criopreservação difundiu-se rapidamente para se tornar um componente essencial da prática de humanos e animais de reprodução assistida e na conservação dos recursos genéticos animais. Atualmente, existem dois principais métodos utilizados para criopreservar oócitos e embriões: congelamento lento e vitrificação. Uma grande variedade de abordagens têm sido usadas para tentar melhorar as técnicas e milhões de animais e milhares de crianças que nasceram a partir de embriões criopreservados. No entanto, subsistem deficiências significativas associadas à criopreservação ainda precisam ser superadas, uma vez que a formação de cristais de gelo, os efeitos da solução e choque osmótico parecem causar cryoinjuries vários pós-descongelamento de oócitos e embriões. Congelamento lento com freezers programável tem a vantagem de utilizar baixas concentrações de crioprotetores, que são geralmente associados com toxicidade química e choque osmótico, mas a sua capacidade para evitar a formação de cristais de gelo em baixas concentrações é limitado. Congelamento lento também induz efeitos super-resfriamento que deve ser evitado o uso de semeadura manual ou automático<sup&gt; 4</sup&gt;. No processo de vitrificação, altas concentrações de crioprotetores inibir a formação de cristais de gelo e levar à formação de um estado vitrificado vitrificados em que a água se solidifica, mas não expandido. No entanto, devido à toxicidade do cyroprotectants nas concentrações utilizadas, os oócitos / embriões só podem ser expostos à solução crioprotetora por um período muito curto de tempo e em uma solução de volume mínimo, antes de submergir as amostras diretamente em nitrogênio líquido<sup&gt; 5</sup&gt;. Na última década, a vitrificação tem se tornado mais popular porque é um método muito rápido em que nenhum equipamento caro (freezer programável) é necessária. No entanto, o congelamento lento continua a ser o método mais amplamente utilizado para oócito / embrião criopreservação. Neste vídeo-artigo vamos mostrar, passo-a-passo, como coletar e congelar lentamente oócitos de hamster com altas taxas de sobrevivência pós-descongelamento. O mesmo procedimento também pode ser aplicado com sucesso congelar e descongelar embriões de camundongos em diferentes estágios de desenvolvimento pré-implantação.

Watch this Scientific Journal Video about Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos at JoVE.com

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Neste vídeo-artigo apresentamos uma demonstração passo-a-passo sobre como coletar e criopreservar oócitos de hamster com altas taxas de sobrevivência pós-descongelamento. O mesmo procedimento também pode ser aplicado com sucesso congelar e descongelar embriões de camundongos em diferentes estágios de desenvolvimento pré-implantação.

Recolha e criopreservação de óvulos e embriões de camundongos Hamster

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

collection-and-cryopreservation-of-hamster-oocytes-and-mouse-embryos

Research

JoVE Journal

Biology

16.9K visualizações.  Universitat Autonoma de Barcelona. Neste vídeo-artigo apresentamos uma demonstração passo-a-passo sobre como coletar e criopreservar oócitos de hamster com altas taxas de sobrevivência pós-descongelamento. O mesmo procedimento também pode ser aplicado com sucesso congelar e descongelar embriões de camundongos em diferentes estágios de desenvolvimento pré-implantação.

Vídeo: Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

Recuperação de oócitos de camundongos

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

Biologia

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Derivação das linhas de células-tronco de embriões pré-implantação do rato

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Biologia do Desenvolvimento

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

Uso de embriões congelados para produção de ratos geneticamente modificados

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

Genética

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

Vitrificação de Volume Mínimo de Oócitos Felinos Imaturos

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Avaliação do mouse microinjeção de oócitos, maturação e ploidia

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

Criopreservação de embriões de camundongos por Vitrificação Etileno Glicol-Based

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Criopreservação do mouse Sperm and Recovery utilizando o I · Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Rato Desenvolvimento Embrionário em todo um sistema de cultura de embriões sem soro

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Meiotic Avaliação de fuso em oócitos mouse por silenciamento mediado por siRNA

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

Criopreservação de Zebrafish espermatogónias por toda agulha testículos imersos refrigeração ultra rápida

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...