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

Questo lavoro è stato sostenuto dal Ministerio de Educación y spagnolo Ciencia (BIO 2006-11792) e la Generalitat de Catalunya (2005-SGR00437). Gli autori ringraziano Marc Puigcerver e Jonatan Lucas per la loro assistenza tecnica per la preparazione dei mezzi. Tutto il personale da d&#39;Servei Estabulari è riconosciuto per la stabulazione degli animali, e soprattutto Juan Jose Martin e Javier Benito per il loro contributo supplementare a registrare parte di questo video. BCN è un collega del portoghese Fundação para a Ciência e Tecnologia e SG è un compagno dello spagnolo Ministerio de Educación y Ciencia.

La cura degli animali e le procedure sono state condotte secondo le linee guida e regolamenti approvati dal Comitato Etico per animali e la ricerca umana della Universitat Autònoma di Barcellona, ​​in Spagna. I. ovociti collezione <ol><li> Controllare e selezionare criceti femmina (Mesocricetus auratus; 8-12 settimana di età) del ceppo d&#39;oro siriano per la presenza di uno scarico di spessore vaginale (scarica fase postovulatoria). </li><li> Indurre femmine selezionati per superovulate mediante iniezione intraperitoneale di 40 UI di PMSG (Pregnant Mare Serum gonadotropina), seguita da 40 UI di hCG (gonadotropina corionica umana) 60 ore dopo. </li><li> Euthanize criceti femmina 16 ore dopo la somministrazione di hCG in una camera di anidride carbonica. </li><li> Isolare ovidotti dalle femmine, come mostrato nel video e trasferirli su una goccia di KSOM-H media. </li><li> Raccogliere ovocita-cumulo complessi strappando l&#39;ampolla ovidotto sotto un microscopio stereoscopico in una goccia di ialuronidasi (156 U / ml) a 37 ° C. Ovociti sarà liberato da cellule del cumulo in circa 5 minuti. </li><li> Pick up ovociti da ialuronidasi e lavarli due volte in KSOM-H media. </li></ol> Per la raccolta di embrioni di topo, topi di sesso femminile (Mus musculus, 6-8 settimane di età) sono indotti a superovulate mediante iniezione intraperitoneale di 5 UI di PMSG seguito da 5 UI di hCG dopo 48 ore e accoppiati con topi di sesso maschile. Le femmine sono sacrificati per dislocazione cervicale a 24-26, 44-46 o 54-56 ore dopo la somministrazione di hCG per ottenimento dei pronuclei, 2-4-cell o cellule di embrioni fase, rispettivamente. Raccolta di embrioni pronuclei viene eseguita esattamente come descritto per ovociti di criceto. Embrioni nelle fasi di 2-celle e 4-celle vengono raccolti svuotando la ovidotti con KSOM-H media [6]. Nota: A seguito di questo protocollo circa 20 o 30 ovociti / embrioni possono essere raccolti da ogni femmina. II. Preparazione di gelo e disgelo soluzioni <ol><li> Preparare la soluzione mi diluendo 0,57 ml di propilenglycol in 5 ml di KSOM-H (1,5 M di propilenglycol). </li><li> Preparare la soluzione disgelo con l&#39;aggiunta di 2 ml di KSOM-H ad un tubo con 0,2052 g di saccarosio (0,3 M di saccarosio). </li><li> Preparare la soluzione di congelamento con l&#39;aggiunta di 2 ml di soluzione che ho ad un tubo con 0,0684 g di saccarosio (1,5 M di saccarosio propilenglycol e 0,1 M). </li></ol> Nota: I Solution e il congelamento e scongelamento soluzioni devono essere preparate poco prima del loro utilizzo. Tuttavia, la determinata quantità di saccarosio da utilizzare per la preparazione delle soluzioni di congelamento e scongelamento può essere ponderata in anticipo e conservati in 3 ml tubi a temperatura ambiente per diversi mesi. III. Protocollo di congelamento <ol><li> Ovociti trasferimento / embrioni dal KSOM-H medio-una goccia di soluzione di I e incubare per 7-15 minuti a temperatura ambiente. </li><li> Nel frattempo, preparare un piatto di 90 millimetri di Petri per caricare il cannucce mettendo 90 gocce ml di congelamento e scongelamento soluzioni come viene mostrato nel video (1 goccia di ogni soluzione / paglia). </li><li> Trasferire il numero desiderato di ovociti / embrioni per le gocce soluzione di congelamento. Nota: Di solito trasferimento tra 5-30 ovociti o embrioni / drop. </li><li> Allegare una cannuccia in un sistema controllato aspirazione bocca e caricarlo seguendo questo ordine: soluzione che, d&#39;aria, con la caduta di congelamento degli ovociti, aria, calo di scongelamento, aria. </li><li> Una volta la paglia viene caricato, mantenere l&#39;aspirazione fino a quando la prima goccia introdotto (soluzione che ho) raggiunge il polimero alla fine superiore della paglia e tenute questo fine. Thermo-seal l&#39;altra estremità della paglia o di tenuta con un tappo in plastica. </li><li> Accendere il congelatore programmabile e selezionare il programma di congelamento. Il programma di congelamento utilizzato in questo protocollo si basa sulla velocità di raffreddamento originariamente pubblicato da Lassalle et al. [7]: <ul><li> 2 ° C / min dalla temperatura ambiente a -7 ° C 0 ° C / min per 10 minuti per consentire l&#39;equilibrio di temperatura e di induzione di semina (vedi sotto) </li><li> 0,3 ° C / min da -7 ° C a -30 ° C 0 ° C / min per 2 minuti per consentire l&#39;equilibrio di temperatura </li><li> 35 ° C / min da -30 ° C a -130 ° C </li></ul></li><li> Caricare le cannucce sugli slot del titolare del congelatore programmabile, assicurandosi che la parte della paglia con la goccia di soluzione di scongelamento è rivolto verso l&#39;esterno. Avviare il programma di congelamento. </li><li> Quando la temperatura del campione raggiunge i -7 ° C ed il raffreddamento è in attesa, effettuare la semina manuale: tuffo la pinza in azoto liquido, tirare i titolari un po &#39;fuori dal freezer per esporre la parte del cannucce contenente le gocce di soluzione di scongelamento, e subito tocco questa parte del cannucce con le pinze. Ripetere per ogni titolare e ogni paglia nel supporto, e poi lasciare che il programma di congelamento fino alla fine. </li><li> Quando il programma è completato, i titolari tuffo in azoto liquido. </li><li> Rimuovere le cannucce daltitolari e loro trasferimento in un alberello con etichetta. </li><li> Infine, memorizzare i alberello all&#39;interno di un serbatoio di azoto liquido. </li></ol> IV. Scongelamento protocollo <ol><li> Rimuovere la cannuccia dal serbatoio di azoto liquido. </li><li> Mantenere la paglia a temperatura ambiente per 40 secondi. </li><li> Mettere a bagno la paglia in un bagno d&#39;acqua a 30 ° C durante 40 secondi. </li><li> Togliere la spina dalla paglia o tagliare il termosaldata punta. </li><li> Svuotare la paglia in un 35-mm piastra di Petri e mescolare delicatamente le due gocce contenenti le soluzioni di congelamento e scongelamento. </li><li> Lascia ovociti / embrioni nella miscela per 15 minuti a temperatura ambiente. </li><li> Trasferimento ovociti / embrioni di un calo fresco di KSOM-H per altri 15 minuti a 37 ° C. </li><li> Ovociti / embrioni sono ora pronti per ulteriori manipolazioni. </li></ol> Nota: Durante i passi 6 e 7 si dovrebbe vedere che gli ovociti scongelati / embrioni lentamente si gonfiano a causa di reidratazione. V. Rappresentante Risultati L&#39;utilizzo del protocollo di crioconservazione qui riportati si traduce in un tasso di sopravvivenza di circa il 95% per ovociti di criceto e del&gt; 90%, 85% e 80% per il mouse B6CBAF1 embrioni al pronucleo, 2-cell stadi e 4-celle, rispettivamente. Quando sono embrioni di topo coltivati ​​in vitro in KSOM dopo lo scongelamento, almeno 70 al 80% dovrebbe arrivare allo stadio di blastocisti in condizioni di coltura ottimali. Si raccomanda di includere un gruppo di controllo (paglia) di ovociti / embrioni in ogni esperimento di congelamento e di scongelamento questo gruppo prima del resto degli ovociti / embrioni vengono utilizzati per ulteriori manipolazioni. Se i tassi di sopravvivenza per il gruppo di controllo sono inferiori alle attese, disgelo un&#39;altra paglia dello stesso lotto. Se i tassi di sopravvivenza per questo secondo gruppo sono bassi, scartare il restante cannucce da questo lotto di 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>

Con questo protocollo e ovociti di criceto embrioni di topo possono essere crioconservati con successo. Una volta congelati, gli ovociti / embrioni possono essere conservati in serbatoi di azoto liquido a tempo indeterminato e recuperato in qualsiasi momento o luogo desiderato. Questo offre il vantaggio di avere campioni quasi pronto per l&#39;uso in caso di necessità. D&#39;altra parte, questo protocollo può essere utilizzato anche per generare criobanche embrione di ceppi di topi di valore (come linee transgeniche), come alternativa eco...

<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

Embrioni e ovociti crioconservati con successo sono stati più di 30 anni fa, quando Whittingham<i&gt; Et al.</i&gt;<sup&gt; 1</sup&gt; E Wilmut<sup&gt; 2</sup&gt; Separatamente descritto embrioni di topo che potrebbero essere congelati e conservati a -196 ° C e, pochi anni dopo, Parkening<i&gt; Et al</i&gt;.<sup&gt; 3</sup&gt; Segnalato la nascita di piccoli vivi da fecondazione in vitro (IVF) di ovociti crioconservati. Da allora, l&#39;uso di tecniche di crioconservazione è rapidamente diffusa a diventare una componente essenziale nella pratica della umana e animale riproduzione assistita e nella conservazione delle risorse genetiche animali. Attualmente, ci sono due metodi principali utilizzati per crioconservare gli ovociti e gli embrioni: il congelamento lento e vitrificazione. Una vasta gamma di approcci sono stati utilizzati per cercare di migliorare sia le tecniche e milioni di animali e migliaia di bambini sono nati da embrioni crioconservati. Tuttavia, le carenze importanti associati alla crioconservazione devono ancora essere superate, in quanto formazione di cristalli di ghiaccio, effetti soluzione e shock osmotico sembrano causare cryoinjuries diversi post-scongelamento degli ovociti e degli embrioni. Congelamento lento con congelatori programmabili ha il vantaggio di usare basse concentrazioni di crioprotettori, che sono di solito associati a tossicità chimica e shock osmotico, ma la loro capacità di evitare la formazione di cristalli di ghiaccio a basse concentrazioni è limitata. Congelamento lento induce anche gli effetti surraffreddamento che deve essere evitato di usare la semina manuale o automatico<sup&gt; 4</sup&gt;. Nel processo di vetrificazione, alte concentrazioni di crioprotettori inibire la formazione di cristalli di ghiaccio e portare alla formazione di uno stato simile a vetro ceramico in cui è solidificato acqua, ma non ampliato. Tuttavia, a causa della tossicità del cyroprotectants alle concentrazioni utilizzate, gli ovociti / embrioni non può che essere esposti alla soluzione crioprotettore per un periodo di tempo molto breve e in una soluzione di volume minimo, prima sommergendo i campioni direttamente in azoto liquido<sup&gt; 5</sup&gt;. Negli ultimi dieci anni, vetrificazione è diventato più popolare perché è un metodo molto veloce in cui è richiesta nessuna attrezzatura costosa (congelatore programmabile). Tuttavia, il congelamento lento continua ad essere il metodo più utilizzato per ovocita / embrione crioconservazione. In questo video-articolo mostriamo, passo per passo, come raccogliere e lentamente congelare ovociti di criceto con elevate post-disgelo tassi di sopravvivenza. La stessa procedura può anche essere applicato con successo per congelare e scongelare embrioni di topo a diversi stadi di sviluppo preimpianto.

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

In questo video-articolo vi presentiamo un passo-passo dimostrazione su come raccogliere e crioconservare gli ovociti di criceto con elevate post-disgelo tassi di sopravvivenza. La stessa procedura può anche essere applicato con successo per congelare e scongelare embrioni di topo a diversi stadi di sviluppo preimpianto.

Raccolta e Crioconservazione degli ovociti e di embrioni di topo criceto

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 Views.  Universitat Autonoma de Barcelona. In questo video-articolo vi presentiamo un passo-passo dimostrazione su come raccogliere e crioconservare gli ovociti di criceto con elevate post-disgelo tassi di sopravvivenza. La stessa procedura può anche essere applicato con successo per congelare e scongelare embrioni di topo a diversi stadi di sviluppo preimpianto.

Video: 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.

Recupero degli ovociti mouse

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

Ricerca

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.

Derivazione di linee di cellule staminali da embrioni di Preimplantation Mouse

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

Biologia dello sviluppo

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 di embrioni congelati per la produzione ad alta efficienza di topi geneticamente modificati

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

Genetica

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.

Vitrificazione del volume minimo degli ovociti felini immaturi

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.

Microiniezione degli ovociti mouse, Maturazione e Ploidia valutazione

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.

Crioconservazione di embrioni di topo da glicole etilenico-Based Vetrificazione

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.

Crioconservazione del mouse Sperm and Recovery utilizzando il Kit · I Cryo

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.

Embrionali di topo di sviluppo in un embrione di tutta la cultura di sistema privo di siero

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 mandrino valutazione in mouse Ovociti mettendo a tacere siRNA-mediata

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.

Crioconservazione degli spermatogoni di Zebrafish dall'intero testicoli ago immerso di raffreddamento ultra-rapido

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 ...