The authors received no specific funding for this work. Professor Maria Grazia Cappai and Dr. Valeria Pasciu are gratefully acknowledged for the video voiceover and for setting up the lab during the video making.

The animal protocol and the implemented procedures described below are in accordance with the ethical guidelines in force at the University of Sassari, in compliance with the European Union Directive 86/609/EC and the recommendation of the Commission of the European Communities 2007/526/EC.
1. Preparation of media for oocyte manipulation
<ol>
	<li>Prepare the medium for transport of collected ovaries by supplementing Dulbecco&#39;s phosphate buffered saline with 0.1 g/L penicillin and 0.1 g/...

<ol>
	<li>Arav, A. <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+oocytes+and+embryos.">Cryopreservation of oocytes and embryos.</a> Theriogenology. 81 (1), 96-102 (2014).</li><li>Mullen, S. F., Fahy, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chronologic+review+of+mature+oocyte+vitrification+research+in+cattle,+pigs,+and+sheep.">A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep.</a> Theriogenology. 78 (8), 1709-1719 (2012).</li><li>Hwang, I. S., Hochi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+cryopreservation+of+bovine+oocytes.">Recent progress in cryopreservation of bovine oocytes.</a> BioMed Research International. 2014, (2014).</li><li>Berlinguer, F., et al. <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+trehalose+co-incubation+on+in+vitro+matured+prepubertal+ovine+oocyte+vitrification.">Effects of trehalose co-incubation on in vitro matured prepubertal ovine oocyte vitrification.</a> Cryobiology. 55 (1), (2007).</li><li>Quan, G., Wu, G., Hong, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+Cryopreservation+Based+in+Sheep:+The+Current+Status+and+Future+Perspective.">Oocyte Cryopreservation Based in Sheep: The Current Status and Future Perspective.</a> Biopreservation and Biobanking. 15 (6), 535-547 (2017).</li><li>Succu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recovery+time+after+warming+restores+mitochondrial+function+and+improves+developmental+competence+of+vitrified+ovine+oocytes.">A recovery time after warming restores mitochondrial function and improves developmental competence of vitrified ovine oocytes.</a> Theriogenology. 110, (2018).</li><li>Succu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+of+in+vitro+matured+ovine+oocytes+affects+in+vitro+pre-implantation+development+and+mRNA+abundance.">Vitrification of in vitro matured ovine oocytes affects in vitro pre-implantation development and mRNA abundance.</a> Molecular Reproduction and Development. 75 (3), (2008).</li><li>Succu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+Devices+Affect+Structural+and+Molecular+Status+of+In+Vitro+Matured+Ovine+Oocytes.">Vitrification Devices Affect Structural and Molecular Status of In Vitro Matured Ovine Oocytes.</a> Molecular Reproduction and Development. 74, 1337-1344 (2007).</li><li>Hosseini, S. M., Asgari, V., Hajian, M., Nasr-Esfahani, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic,+rather+than+nuclear-DNA,+insufficiencies+as+the+major+cause+of+poor+competence+of+vitrified+oocytes.">Cytoplasmic, rather than nuclear-DNA, insufficiencies as the major cause of poor competence of vitrified oocytes.</a> Reproductive BioMedicine Online. , (2015).</li><li>Succu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+concentration+in+vitrification+medium+affects+the+developmental+competence+of+in+vitro+matured+ovine+oocytes.">Calcium concentration in vitrification medium affects the developmental competence of in vitro matured ovine oocytes.</a> Theriogenology. 75 (4), (2011).</li><li>Sanaei, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+vitrification+of+in+vitro+matured+ovine+oocytes;+beneficial+effects+of+Ethylene+Glycol+Tetraacetic+acid,+an+intracellular+calcium+chelator.">An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid, an intracellular calcium chelator.</a> Cryobiology. 84, 82-90 (2018).</li><li>Quan, G. B., Wu, G. Q., Wang, Y. J., Ma, Y., Lv, C. R., Hong, Q. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+maturation+and+developmental+capability+of+ovine+oocytes+at+germinal+vesicle+stage+following+vitrification+using+different+cryodevices.">Meiotic maturation and developmental capability of ovine oocytes at germinal vesicle stage following vitrification using different cryodevices.</a> Cryobiology. 72 (1), 33-40 (2016).</li><li>Fern&#225;ndez-Reyez, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=maturation+and+embryo+development+in+vitro+of+immature+porcine+and+ovine+oocytes+vitrified+in+different+devices.">maturation and embryo development in vitro of immature porcine and ovine oocytes vitrified in different devices.</a> Cryobiology. 64 (3), 261-266 (2012).</li><li>Ahmadi, E., Shirazi, A., Shams-Esfandabadi, N., Nazari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidants+and+glycine+can+improve+the+developmental+competence+of+vitrified/warmed+ovine+immature+oocytes.">Antioxidants and glycine can improve the developmental competence of vitrified/warmed ovine immature oocytes.</a> Reproduction in Domestic Animals. 54 (3), 595-603 (2019).</li><li>Barrera, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+delipidated+estrous+sheep+serum+supplementation+on+in+vitro+maturation,+cryotolerance+and+endoplasmic+reticulum+stress+gene+expression+of+sheep+oocytes.">Impact of delipidated estrous sheep serum supplementation on in vitro maturation, cryotolerance and endoplasmic reticulum stress gene expression of sheep oocytes.</a> PLoS ONE. 13 (6), (2018).</li><li>Walker, S. K., Hill, J. L., Kleemann, D. O., Nancarrow, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Ovine+Embryos+in+Synthetic+Oviductal+Fluid+Containing+Amino+Acids+at+Oviductal+Fluid+Concentrations.">Development of Ovine Embryos in Synthetic Oviductal Fluid Containing Amino Acids at Oviductal Fluid Concentrations.</a> Biology of Reproduction. 55 (3), 703-708 (1996).</li><li>Kuwayama, M., Vajta, G., Kato, O., Leibo, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+vitrification+method+for+cryopreservation+of+human+oocytes.">Highly efficient vitrification method for cryopreservation of human oocytes.</a> Reproductive BioMedicine Online. 11 (3), 300-308 (2005).</li><li>Wu, X., Jin, X., Wang, Y., Mei, Q., Li, J., Shi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+spectral+properties+of+novel+chlorinated+pH+fluorescent+probes.">Synthesis and spectral properties of novel chlorinated pH fluorescent probes.</a> Journal of Luminescence. 131 (4), 776-780 (2011).</li><li>Dell'Aquila, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prooxidant+effects+of+verbascoside,+a+bioactive+compound+from+olive+oil+mill+wastewater,+on+in+vitro+developmental+potential+of+ovine+prepubertal+oocytes+and+bioenergetic/oxidative+stress+parameters+of+fresh+and+vitrified+oocytes.">Prooxidant effects of verbascoside, a bioactive compound from olive oil mill wastewater, on in vitro developmental potential of ovine prepubertal oocytes and bioenergetic/oxidative stress parameters of fresh and vitrified oocytes.</a> BioMed Research International. 2014, (2014).</li><li>Gadau, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+quantitative+analysis+on+&#945;-tubulin+modifications+in+glioblastoma+cells.">Morphological and quantitative analysis on &#945;-tubulin modifications in glioblastoma cells.</a> Neuroscience Letters. 687, 111-118 (2018).</li><li>los Reyes, M. D., Palomino, J., Parraguez, V. H., Hidalgo, M., Saffie, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+distribution+and+meiotic+progression+in+canine+oocytes+during+in+vivo+and+in+vitro+maturation.">Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation.</a> Theriogenology. , (2011).</li><li>Leoni, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+kinetic+of+the+first+meiotic+division+and+in+active+mitochondrial+distribution+between+prepubertal+and+adult+oocytes+mirror+differences+in+their+developmental+competence+in+a+sheep+model.">Differences in the kinetic of the first meiotic division and in active mitochondrial distribution between prepubertal and adult oocytes mirror differences in their developmental competence in a sheep model.</a> PLoS ONE. 10 (4), (2015).</li><li>Berlinguer, F., et al. <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+trehalose+co-incubation+on+in+vitro+matured+prepubertal+ovine+oocyte+vitrification.">Effects of trehalose co-incubation on in vitro matured prepubertal ovine oocyte vitrification.</a> Cryobiology. 55 (1), 27-34 (2007).</li><li>Serra, E., Gadau, S. D., Berlinguer, F., Naitana, S., Succu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+features+and+microtubular+changes+in+vitrified+ovine+oocytes.">Morphological features and microtubular changes in vitrified ovine oocytes.</a> Theriogenology. 148, 216-224 (2020).</li><li>Asgari, V., Hosseini, S. M., Ostadhosseini, S., Hajian, M., Nasr-Esfahani, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+dependent+effect+of+post+warming+interval+on+microtubule+organization,+meiotic+status,+and+parthenogenetic+activation+of+vitrified+in+vitro+matured+sheep+oocytes.">Time dependent effect of post warming interval on microtubule organization, meiotic status, and parthenogenetic activation of vitrified in vitro matured sheep oocytes.</a> Theriogenology. 75 (5), 904-910 (2011).</li><li>Ciotti, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+spindle+recovery+is+faster+in+vitrification+of+human+oocytes+compared+to+slow+freezing.">Meiotic spindle recovery is faster in vitrification of human oocytes compared to slow freezing.</a> Fertility and Sterility. 91 (6), 2399-2407 (2009).</li><li>Ledda, S., Bogliolo, L., Leoni, G., Naitana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Coupling+and+Maturation-Promoting+Factor+Activity+in+In+Vitro-Matured+Prepubertal+and+Adult+Sheep+Oocytes1.">Cell Coupling and Maturation-Promoting Factor Activity in In Vitro-Matured Prepubertal and Adult Sheep Oocytes1.</a> Biology of Reproduction. 65 (1), 247-252 (2001).</li><li>Palmerini, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+maturation+is+slowed+in+prepubertal+lamb+oocytes:+ultrastructural+evidences.">In vitro maturation is slowed in prepubertal lamb oocytes: ultrastructural evidences.</a> Reproductive Biology and Endocrinology. 12, (2014).</li><li>Leoni, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relations+between+relative+mRNA+abundance+and+developmental+competence+of+ovine+oocytes.">Relations between relative mRNA abundance and developmental competence of ovine oocytes.</a> Molecular Reproduction and Development. 74 (2), 249-257 (2007).</li><li>Succu, S., et al. <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+vitrification+solutions+and+cooling+upon+in+vitro+matured+prepubertal+ovine+oocytes.">Effect of vitrification solutions and cooling upon in vitro matured prepubertal ovine oocytes.</a> Theriogenology. 68 (1), 107-114 (2007).</li><li>Larman, M. G., Sheehan, C. B., Gardner, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-free+vitrification+reduces+cryoprotectant-induced+zona+pellucida+hardening+and+increases+fertilization+rates+in+mouse+oocytes.">Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes.</a> Reproduction. 131 (1), 53-61 (2006).</li><li>Yeste, M., Jones, C., Amdani, S. N., Patel, S., Coward, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+activation+deficiency:+a+role+for+an+oocyte+contribution.">Oocyte activation deficiency: a role for an oocyte contribution.</a> Human Reproduction Update. 22 (1), 23-47 (2016).</li><li>Rienzi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte,+embryo+and+blastocyst+cryopreservation+in+ART:+systematic+review+and+meta-analysis+comparing+slow-freezing+versus+vitrification+to+produce+evidence+for+the+development+of+global+guidance.">Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance.</a> Human Reproduction Update. 23 (2), 139-155 (2017).</li><li>De Santis, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+vitrification:+influence+of+operator+and+learning+time+on+survival+and+development+parameters.">Oocyte vitrification: influence of operator and learning time on survival and development parameters.</a> Placenta. 32, 280-281 (2011).</li><li>Zhang, X., Catalano, P. N., Gurkan, U. A., Khimji, I., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+technologies+in+medical+applications+of+minimum+volume+vitrification.">Emerging technologies in medical applications of minimum volume vitrification.</a> Nanomedicine. 6 (6), 1115-1129 (2011).</li></ol>

Oocyte cryopreservation in domestic animals can allow not only the long-term conservation of female genetic resources, but also advance the development of embryonic biotechnologies. Thus, the development of a standard method for oocyte vitrification would advantage both the livestock and the research sector. In this protocol, a complete method for adult sheep oocyte vitrification is presented and could represent a solid starting point for the development of an efficient vitrification system for juvenile oocyte.
<p cl...

Long-term storage of the female gametes can offer a wide range of applications, such as improving domestic animal breeding by genetic selection programs, contributing to preserve biodiversity through the ex-situ wildlife species conservation program, and boosting in vitro biotechnology research and applications thanks to the availability of stored oocytes to be incorporated in in vitro embryo production or nuclear transplantation programs1,2,3. Juvenile oocyte vitrification would also increase genetic gain by shortening the generation interval in breeding programs4. Vitrification by ultra-rapid cooling and warming of oocytes is currently considered a standard approach for livestock oocytes cryopreservation5. In ruminants, before vitrification, oocytes are usually matured in vitro, after retrieval from follicles obtained from abattoir-derived ovaries2. Adult, and especially prepubertal ovaries4,6, can indeed supply a virtually unlimited number of oocytes to be cryopreserved.
In cattle, after oocyte vitrification and warming, blastocyst yields at &#62;10% have been commonly reported by several laboratories during the last decade3. However, in small ruminants oocyte vitrification is still considered relatively new for both juvenile and adult oocytes, and a standard method for sheep oocyte vitrification remains to be established2,5. Despite recent advancements, the vitrified and warmed oocyte indeed presents several functional and structural alterations that limit their developmental potential7,8,9. Thus, few articles have reported blastocyst development at 10% or more in vitrified/warmed sheep oocytes2. Several approaches have been investigated to reduce the above-mentioned alterations: optimizing the composition of the vitrification and thawing solutions10,11; experimenting with the use of different cryo-devices8,12,13; and applying specific treatments during in vitro maturation (IVM)4,14,15 and/or during the recovery time after warming6.
Here we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming culture period.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2&#8242;,7&#8242;-Dichlorofluorescin diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>D-6883</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin bovine fraction V, protease free</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>Bisbenzimide H 33342 trihydrochloride (Hoechst 33342)</td>
			<td>Sigma-Aldrich</td>
			<td>14533</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2 2H20)</td>
			<td>Sigma-Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C2404</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica Microsystems GmbH,Wetzlar</td>
			<td>TCS SP5 DMI 6000CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotop Kitazato</td>
			<td>Medical Biological Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteamine</td>
			<td>Sigma-Aldrich</td>
			<td>M9768</td>
			<td></td>
		</tr>
		<tr>
			<td>D- (-) Fructose</td>
			<td>Sigma-Aldrich</td>
			<td>F0127</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)Trehalose dehydrate</td>
			<td>Sigma-Aldrich</td>
			<td>T0167</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg yolk</td>
			<td>Sigma-Aldrich</td>
			<td>P3556</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol (EG)</td>
			<td>Sigma-Aldrich</td>
			<td>324558</td>
			<td></td>
		</tr>
		<tr>
			<td>FSH</td>
			<td>Sigma-Aldrich</td>
			<td>F4021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>G5638</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8790</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H4149</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoutarine</td>
			<td>Sigma-Aldrich</td>
			<td>H1384</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Diaphot, Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Alanine</td>
			<td>Sigma-Aldrich</td>
			<td>A3534</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>Sigma-Aldrich</td>
			<td>A3784</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Asparagine</td>
			<td>Sigma-Aldrich</td>
			<td>A4284</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4534</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cystine</td>
			<td>Sigma-Aldrich</td>
			<td>C8786</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G3126</td>
			<td></td>
		</tr>
		<tr>
			<td>LH</td>
			<td>Sigma-Aldrich</td>
			<td>L6420</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Sigma-Aldrich</td>
			<td>H9511</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Isoleucine</td>
			<td>Sigma-Aldrich</td>
			<td>I7383</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Leucine</td>
			<td>Sigma-Aldrich</td>
			<td>L1512</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L1137</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Sigma-Aldrich</td>
			<td>M2893</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ornithine</td>
			<td>Sigma-Aldrich</td>
			<td>O6503</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Sigma-Aldrich</td>
			<td>P5030</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Sigma-Aldrich</td>
			<td>P4655</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Serine</td>
			<td>Sigma-Aldrich</td>
			<td>S5511</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine</td>
			<td>Sigma-Aldrich</td>
			<td>T1020</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Valine</td>
			<td>Sigma-Aldrich</td>
			<td>V6504</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride heptahydrate (MgSO4.7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Makler Counting Chamber</td>
			<td>Sefi-Medical Instruments ltd.Biosigma S.r.l.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma-Aldrich</td>
			<td>M5017</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Red CM-H2XRos</td>
			<td>ThermoFisher</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>New born calf serum heat inactivated (FCS)</td>
			<td>Sigma-Aldrich</td>
			<td>N4762</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma-Aldrich</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl alcohol (87-90% hydrolyzed, average mol wt 30,000-70,000)</td>
			<td>Sigma-Aldrich</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Sigma-Aldrich</td>
			<td>S2263</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2202</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dl-lactate solution syrup</td>
			<td>Sigma-Aldrich</td>
			<td>L4263</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256</td>
			<td></td>
		</tr>
		<tr>
			<td>Sperm Class Analyzer</td>
			<td>Microptic S.L.</td>
			<td>S.C.A. v 3.2.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Statistical software Minitab 18.1</td>
			<td>2017 Minitab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Olimpus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>S9137</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T7146</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Sigma-Aldrich</td>
			<td>15,456-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

vitrification of in vitro matured oocytes collected from adult and prepubertal ovaries in sheep

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. 
Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

The cryotolerance of oocyte from juvenile donors is lower compared to adult ones. The first effect observed is a lower post-warming survival rate compared to adult oocytes (Figure 1A; &#967;2 test P&#60;0.001). Juvenile oocytes showed a lower membrane integrity after warming (Figure 1B). The use of trehalose in the maturation medium was intended to verify whether this sugar could reduce cryoinjuries in juvenile oocytes. The data have demonstrated<sup ...

Watch this Scientific Journal Video about Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep at JoVE.com

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily ...

The maintaining of oocyte potential viability after longterm storage represent a tool of great opportunity, as it would improve domestic animal breeding by genetic selection programs, contribute to preserve biodiversity through wildlife species conservation, and increase the research in biotechnology. Juvenile oocyte vitrification was a lot shorter in the generation interval in breeding programs. Vitrifaction by ultrarapid cooling and warming is considered a standard approach in cattle oocyte preservation.However, in sheep this procedure is still considered relatively new and a standard method is lacking. In this video, we describe a protocol for the vitrification of sheep oocyte, collected from both juvenile and adult donors, and in vitro matured to prior to cryopreservation. The protocol include all the procedures from in vitro maturation, vitrification, warming, and post-warming culture.After recovering the Cumulus oocytes complex, where matured in vitro for 24 hours in static a condition that include for juvenile oocyte, the supplementation with hundred micromolar of trehalose in maturation medium. Following in vitro maturation, the oocytes were denuded of Cumulus cells mechanically by gently pipetting and examined under stereo microscope, with 60x magnification. The selection of the oocytes to submit at the vitrification procedures represent the first important step to warranty the successful application of this procedure.Only those with the uniform cytoplasm, with homogeneous distribution of lipid droplets, integrity of the all Emma wide and continuous pivotal in space, compact zona pellucida the outer diameter of about 19 micrometers and showing the extrusion of the first polar body, and thus at the M2, stage must be selected. Vitrification was performed using the minimum essential volume method, with a Cryotops device. After selection, a group of five juvenile oocytes were conditioned at 38.5 Celsius degrees in base medium for two minutes.The oocytes were then dehydrated by three minutes exposure to a calibration solution. The loading of the oocyte in the vitrification device used is an important and critical step. Cryotop uses a polypropylene strip attached to a holder.In this method, the oocytes in the vitrification solution, less than 0 point milliliter are rapidly loaded with a glass capillary on top of the film strip. Then the solution must be removed, leaving behind a thin layer enough to cover the cells to be cryopreserved. Before being loaded in Cryotop device and directly plunged into liquid nitrogen within 30 seconds.For warm interior biological temperature, the content of each vitrification device was transferred from liquid nitrogen into 200 microliter drops of decreasing trehalose solution. Oocytes were transferred set was, dragging the oocytes with a thin, glass pipette, to promote the removal of intracelular cryoprotectants. Finally, the oocytes were pre-breeded in a base medium in incubator in 5%carbon dioxide.Immediately after warming, and for each time point of post-warming culture, oocytes were morphologically evaluated using an inverted microscope with 100x magnification. The cryo tolerance of oocytes from juvenile donors is lower compared to altered ones with lower membrane integrity after warming. The use of trehalose in maturation medium induced in juvenile oocytes higher membrane integrity, increasing the survival rates after vitrification.However cleavage, fertilization, and developmental rates of juvenile oocytes were not increased by trehalose supplementation. The use of media with calcium concentration equal to 2.2 milligrams deciliter for the vitrification of juvenile oocytes, should higher fertilization rates compared to oocytes vitrified with calcium concentration, but no differences were found enabling production. By comparing post-warming culture on different durations, we showed that after four hours of culture oocyte collected from older twos are able to recover the energetic bonds and microtubular setup, and to restore the developmental competence with higher cleavage and blastocytes waste.Mitochondrial activity was higher in vitrified warm juvenile oocytes after four hours of post-warming culture, compared to other time points. Mitochondrial distribution parting also changed during six hours of post warming culture. Moreover, reactive oxygen species intracellular levels were significantly lower in juvenile oocytes at two hours of post-warming culture compared to 0, 4, and 6 hours.However, and in contrast with what found in other oocytes, the rates of spontaneous parthenogentic activation increased during post-warming culture in juvenile oocytes. One of the main advantages of the proposed method is it includes all the steps from oocyte collection to in-vitro maturation, vitrification, and warming. Moreover it includes a post-warming culture period to allow oocyte recovery from the damages incurred during the vitrification procedure.Choosing the optimum time for fertilization is challenging, and it may impact on the outcome of the vitrification program. It should also be considered that, unlike slow freezing, vitrification is a manual technique, and is thus operator dependent. For this reason, a major challenge is the need for skilled technicians and researchers who take into consideration the operator effect in the evaluation of the outcome of the vitrification program.Further studies from our lab will allow in attempting to standardize oocyte vitrification procedure and selection, and in better tailoring media composition to the needs of the juvenile oocyte. A disregard about the use of culture and character and antioxidant we offer promising opportunities.

vitrification-vitro-matured-oocytes-collected-from-adult-prepubertal

Research

JoVE Journal

Developmental Biology

4.9K vues.  University of Sassari. Le maintien de la viabilité potentielle des ovocytes après le stockage à long terme représente un outil de grande opportunité, car il améliorerait l’élevage d’animaux domestiques par des programmes de sélection génétique, contribuerait à préserver la biodiversité grâce à la conservation des espèces sauvages et augmenterait la recherche en biotechnologie. La vitrification des ovocytes juvéniles était beaucoup plus courte dans l’intervalle de génération dans les progra...

Vidéo: Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

cryopreservation of oocytes retrieved from ovarian tissue to optimize fertility preservation in prepubertal girls and women

Cryopreservation of Oocytes Retrieved from Ovarian Tissue to Optimize Fertility Preservation in Prepubertal Girls and Women

We propose a protocol for fertility preservation in prepubertal girls and women at risk of premature ovarian insufficiency. It combines ovarian tissue freezing and cryopreservation of oocytes retrieved from ovarian tissue. This strategy improves the safety and optimizes the reproductive potential of fertility preservation, maximizing the chance of...

minimum volume vitrification of immature feline oocytes

Minimum Volume Vitrification of Immature Feline Oocytes

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...

accurate follicle enumeration in adult mouse ovaries

Accurate Follicle Enumeration in Adult Mouse Ovaries

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian...

in vitro culture strategy for oocytes from early antral follicle in cattle

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage.

detection of dna double-stranded breaks in mouse oocytes

Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ...

Identifying DNA Damage in Prophase-Arrested Oocytes of Mice

isolation of granulosa cell-free stage i oocytes from zebrafish ovaries

Isolation of Granulosa Cell-Free Stage I Oocytes from Zebrafish Ovaries

vitrification of ovarian cortex tissue to achieve a glassy state of aggregation

Author Spotlight: Advancing Fertility Preservation in Young Female Cancer Patients Through Ovarian Tissue Vitrification and In Vitro Follicular Growth

A protocol for the vitrification of ovarian tissue, as an alternative cryopreservation method to the widely used slow freezing protocol, is...

Follicular Viability Assessment in Vitrified Human Ovarian Cortex Tissues

https://cloudfront.jove.com/CDNSource/teasers/66801_b.jpg

rapid isolation of stage i oocytes in zebrafish devoid of granulosa cells

Author Spotlight: Enhanced Method for Isolating Pure Zebrafish Stage I Oocytes

This protocol describes a modified procedure for rapidly isolating clean stage I oocytes in zebrafish devoid of granulosa cells, thereby providing a convenient method for oocyte-specific...

Improved Procedure to Obtain Granulosa Cell-Free Stage I Zebrafish Oocytes

https://cloudfront.jove.com/CDNSource/teasers/66458_b.jpg

in situ labeling of mitochondrial dna replication in drosophila adult ovaries by edu staining

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously...

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

mouse antral oocyte isolation: a method to isolate antral oocytes from freshly harvested mouse ovaries

This video describes the isolation of antral oocytes from freshly harvested mouse ovaries. These antral oocytes can be used for further characterization and developmental...

Mouse Antral Oocyte Isolation: A Method to Isolate Antral Oocytes from Freshly Harvested Mouse Ovaries

Then, transfer all of the Petri dishes to a 37 degrees Celsius, 5% carbon dioxide incubator to allow the medium to equilibrate. Next, use sterile dissection scissors to make an incision in the skin covering the abdominal wall, followed by an incision in the peritoneum of the first hormone-injected...

collection of germinal vesicle oocytes from the ovaries of cd-1 mice and induction of dna double-strand breaks

Collection of Germinal Vesicle Oocytes from the Ovaries of CD-1 Mice and Induction of DNA Double-Strand Breaks  

transcatheter pulmonary valve replacement from autologous pericardium with a self-expandable nitinol stent in an adult sheep model

Transcatheter Pulmonary Valve Replacement from Autologous Pericardium with a Self-Expandable Nitinol Stent in an Adult Sheep Model

This study demonstrates the feasibility and safety of developing an autologous pulmonary valve for implantation at the native pulmonary valve position by using a self-expandable Nitinol stent in an adult sheep model. This is a step toward developing transcatheter pulmonary valve replacement for patients with right ventricular outflow tract...

separation of follicular cells and oocytes in ovarian follicles of zebrafish

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in...

studying mitochondrial structure and function in drosophila ovaries

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

manipulation and in vitro maturation of xenopus laevis oocytes, followed by intracytoplasmic sperm injection, to study embryonic development

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic...

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

fertility preservation through oocyte vitrification: clinical and laboratory perspectives

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women. Appropriate clinical and laboratory strategies ensure maximum efficacy, efficiency, and safety of fertility preservation...

culture and co-culture of mouse ovaries and ovarian follicles

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian...

analysis of chromosome segregation, histone acetylation, and spindle morphology in horse oocytes

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA...

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Human Blastocyst Biopsy and Vitrification

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow entails a safe and efficient vitrification protocol, due to the turnaround time of the diagnostic techniques and to transfer the selected embryo(s) on a physiological endometrium in a following natural cycle. A biopsy approach encompassing the sequential opening of the zona pellucida and retrieval of 5-10 trophectoderm cells (ideally 7-8) limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions. After proper training, the technique was reproducible across different operators in terms of timing of biopsy (~8 min, ranging 3-22 min based on the number of embryos to biopsy per dish), conclusive diagnoses obtained (~97.5%) and live birth rates after vitrified-warmed euploid blastocyst transfer (&#62;40%). The survival rate after biopsy, vitrification and warming was as high as 99.8%. The re-expansion rate at 1.5 h from warming was as high as 97%, largely dependent on the timing between biopsy and vitrification (ideally &#8804;30 min), blastocyst morphological quality and day of biopsy. In general, it is better to vitrify a collapsed blastocyst; therefore, in non-PGT cycles, laser-assisted artificial shrinkage might be performed to induce embryo collapse prior to cryopreservation. The most promising future perspective is the non-invasive analysis of the IVF culture media after blastocyst culture as a putative source of embryonic DNA. However, this potential avant-garde is still under investigation and a reliable protocol yet needs to be defined and validated.

Blastocyst biopsy and vitrification are required to efficiently conduct preimplantation genetic testing. An approach entailing the sequential opening of the zona pellucida and retrieval of 7-8 trophectoderm cells in day 5-7 post-insemination limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions.

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow ...

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 ...

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary gland is the key regulator of the body's endocrine system. This article describes the development of organoids from the mouse pituitary as a novel 3D in vitro model to study the gland's stem cell population of which the biology and function remain poorly understood.

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...