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

This work was supported by the Spanish Ministerio de Educaci&oacute;n y Ciencia (BIO 2006-11792) and the Generalitat de Catalunya (2005-SGR00437). Authors would like to thank Marc Puigcerver and Jonatan Lucas for their technical assistance in preparing the media. All staff from the Servei d&rsquo;Estabulari is acknowledged for animal housing, and especially Juan Jose Martin and Javier Benito for their additional contribution on recording part of this video. N.C.B. is a fellow of the Portuguese Funda&ccedil;&atilde;o para a Ci&ecirc;ncia e Tecnologia and S.G. is a fellow of the Spanish Ministerio de Educaci&oacute;n y Ciencia.

Animal care and procedures were conducted according to the guidelines and regulations approved by the Ethics Committee on Animal and Human Research of the Universitat Aut&ograve;noma of Barcelona, Spain.
I. Oocyte collection
<ol>
<li>Check and select female hamsters (Mesocricetus auratus; 8-12 week&rsquo;s old) of the Golden Syrian strain for the presence of a thick vaginal discharge (postovulatory discharge).</li>
<li>Induce selected females to superovulate by intraperitoneal injection of 40 IU of PMSG (Pregnant Mare Serum Gonadotropin) followed by 40 IU of hCG (human Chorionic Gonadotropin) 60 hours later.</li>
<li>Euthanize female hamsters 16 hours after administration of hCG in a carbon dioxide chamber.</li>
<li>Isolate oviducts from the females as shown in the video and transfer them to a drop of KSOM-H medium.</li>
<li>Collect oocyte-cumulus complexes by tearing the oviduct ampulla under a stereoscopic microscope in a drop of hyaluronidase (156 U/ml) at 37&deg;C. Oocytes will be freed from cumulus cells in approximately 5 minutes.</li>
<li>Pick up oocytes from hyaluronidase and wash them twice in KSOM-H medium.</li>
</ol>
For the collection of mouse embryos, female mice (Mus musculus; 6-8 week&rsquo;s old) are induced to superovulate by intraperitoneal injection of 5 IU of PMSG followed by 5 IU of hCG 48 hours later and mated with male mice. Females are sacrificed by cervical dislocation at 24-26, 44-46 or 54-56 hours after the administration of hCG for obtention of pronuclear, 2-cell or 4-cell stage embryos, respectively. Collection of pronuclear embryos is performed exactly as described for hamster oocytes. Embryos at the 2-cell and 4-cell stages are collected by flushing the oviducts with KSOM-H medium [6]. Note: Following this protocol approximately 20 to 30 oocytes/embryos can be collected from each female.
II. Preparation of freezing and thawing solutions
<ol>
<li>Prepare solution I by diluting 0.57 ml of propilenglycol in 5 ml of KSOM-H (1.5 M of propilenglycol).</li>
<li>Prepare thawing solution by adding 2 ml of KSOM-H to a tube with 0.2052 g sucrose (0.3 M of sucrose).</li>
<li>Prepare freezing solution by adding 2 ml of solution I to a tube with 0.0684 g sucrose (1.5 M of propilenglycol and 0.1 M sucrose).</li>
</ol>
 Note: Solution I and freezing and thawing solutions must be prepared just before their use. However, the specified amounts of sucrose to be used for the preparation of the freezing and thawing solutions may be weighted in advance and stored in 3 ml-tubes at room temperature for several months.
III. Freezing protocol
<ol>
<li>Transfer oocytes/embryos from the KSOM-H medium to a drop of solution I and incubate for 7-15 minutes at room temperature.</li>
<li>Meanwhile, prepare a 90 mm petri dish to load the straws by placing 90 &micro;l drops of freezing and thawing solutions as it is shown in the video (1 drop of each solution/straw).</li>
<li>Transfer the desired number of oocytes/embryos to the freezing solution drops. 
Note: We usually transfer between 5 to 30 oocytes or embryos/drop.</li>
<li>Attach a straw to a mouth controlled aspiration system and load it following this order: solution I, air, freezing drop with the oocytes, air, thawing drop, air.</li>
<li>Once the straw is loaded, keep aspirating until the first drop introduced (solution I) reaches the polymer at the top end of the straw and seals this end. Thermo-seal the other end of the straw or seal with a plastic plug.</li>
<li>Switch on the programmable freezer and select the freezing program. The freezing program used in this protocol is based on the cooling rates originally published by Lassalle et al. [7]: 
<ul>
<li> 2&deg;C/min from room temperature to -7&deg;C 
0&deg;C/min for 10 min to allow for temperature equilibration and induction of seeding (see below)</li>
<li>0.3&deg;C/min from -7&deg;C to -30&deg;C 
0&deg;C/min for 2 min to allow for temperature equilibration</li>
<li>35&deg;C/min from -30&deg;C to -130&deg;C</li>
</ul>
</li>
<li>Load the straws on the slots of the holder of the programmable freezer, making sure that the part of the straw with the drop of thawing solution is facing outwards. Start the freezing program.</li>
<li>When sample temperature reaches -7&deg;C and the cooling is on hold, perform manual seeding: plunge the forceps into liquid nitrogen, pull the holders slightly out of the freezer to expose the part of the straws containing the drops of thawing solution, and immediately touch this part of the straws with the forceps. Repeat for each holder and each straw in the holder, and then let the freezing program run until the end.</li>
<li>When the program is completed, plunge the holders in liquid nitrogen.</li>
<li>Remove the straws from the holders and transfer them to a labeled gobelet.</li>
<li>Finally, store the gobelet inside a liquid nitrogen tank.</li>
</ol>
IV. Thawing protocol
<ol>
<li>Remove the straw from the liquid nitrogen tank.</li>
<li>Maintain the straw at room temperature for 40 seconds.</li>
<li>Soak the straw in a water bath at 30&deg;C during 40 seconds.</li>
<li>Remove the plug from the straw or cut the thermo-sealed tip.</li>
<li>Empty the straw in a 35-mm petri dish and gently mix the two drops containing the freezing and thawing solutions.</li>
<li>Leave oocytes/embryos in the mixture for 15 minutes at room temperature.</li>
<li>Transfer oocytes/embryos to a fresh drop of KSOM-H for an additional 15 minutes at 37&deg;C.</li>
<li>Oocytes/embryos are now ready for further manipulation.</li>
</ol>
 Note: During steps 6 and 7 you should see that the thawed oocytes/embryos slowly swell up due to rehydration.
V. Representative Results
The use of the cryopreservation protocol reported here results in a survival rate of approximately 95% for hamster oocytes and of &gt;90%, 85% and 80% for mouse B6CBAF1 embryos at the pronuclear, 2-cell and 4-cell stages, respectively. When mouse embryos are cultured in vitro in KSOM after thawing, at least 70 to 80% should reach the blastocyst stage in optimal culture conditions.
It is recommended that you include a control group (straw) of oocytes/embryos in each freezing experiment and that you thaw this group before the rest of the oocytes/embryos are used for further manipulations. If survival rates for the control group are lower than expected, thaw another straw from the same lot. If survival rates for this second group are also low, discard the remaining straws from this freezing lot.

<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>

With this protocol hamster oocytes and mouse embryos can be successfully cryopreserved. Once frozen, oocytes/embryos can be stored in liquid nitrogen tanks indefinitely and recovered at any time or place desired. This offers the advantage of having samples almost ready to use whenever needed. On the other hand, this protocol can also be used to generate embryo cryobanks from valuable mouse strains (such as transgenic lines), as an economical and safer alternative to the maintenance of live animal colonies. It is important to note that this protocol is optimized to cryopreserve hamster oocytes and hybrid B6CBAF1 mouse embryos. Differences in post-thaw survival rates among mouse embryos with different genetic backgrounds have been described 8,9, thus care should be taken to assess cryopreservation conditions on each species or strain of oocytes/embryos and to establish the appropriate protocol before the generation of oocyte or embryo cryobanks.
Hamster oocytes can be used in human assisted reproduction clinics for the sperm penetration test or for sperm chromosome analysis 10, or as practicing material for beginners in intracytoplasmic sperm injection (ICSI). On the other hand, mouse hybrid B6CBAF1 embryos at pronuclear or 2-cell stages can be used in human assisted reproduction centers or in research labs to check the quality of the embryo culture media or the proper functioning of the incubators.

<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

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

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 this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

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

Nanyang Technological University

Research

JoVE Journal

Biology

16.9K Views.  Universitat Autonoma de Barcelona. In deze video-artikel stellen we een stap-voor-stap demonstratie over hoe het verzamelen en hamster eicellen cryopreserve met een hoge post-dooi overleving. Dezelfde procedure kan ook worden toegepast om met succes vorst en dooi muis embryo's in verschillende stadia van pre-implantatie ontwikkeling.

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.

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

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.

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

Developmental Biology

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.

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

Genetics

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.

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.

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.

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.

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.

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.

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.

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 ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

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