Name: cryopreservation of zebrafish spermatogonia by whole testes needle immersed ultra-rapid cooling
Uploaded: 2018-03-04T13:00:00
Description: Watch this Scientific Journal Video about Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling at JoVE.com

Questo studio è stato sostenuto dai nazionali di ricerca, sviluppo e innovazione ufficio d'Ungheria (grant 116912 a MÀY), l'ufficio di costo (cibo e agricoltura costo azione FA1205: AQUAGAMETE), al programma di borse di Hungaricum Stipendium (grant a ZM) e il nuovo Hungarian National eccellenza Predoctoral Fellowship (grant a EK).

Tutti i metodi descritti qui sono stati approvati il diritto ungherese del benessere animale.
1. preparazione dei reagenti
<ol><li>Soluzioni di riserva<ol>
<li>Preparare 1 M trealosio aggiungendo 0,378 g di trealosio diidrato in 1 mL di dH2O. Mix bene fino a quando si scioglie completamente.</li>
		<li>Preparare 1 M saccarosio aggiungendo 0,342 g di saccarosio in 1 mL di dH2O. Mix bene fino a quando si scioglie completamente.</li>
		<li>Preparare 1 M HEPES aggiungendo 0...

<ol>
	<li>Mazur, P., Leibo, S. P., Seidel, G. R. 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+the+germplasm+of+animals+used+in+biological+and+medical+research:+Importance,+impact,+status,+and+future+directions.">Cryopreservation of the germplasm of animals used in biological and medical research: Importance, impact, status, and future directions.</a> Biol. Reprod. 78 (1), 2-12 (2008).</li><li>Carmichael, C., Westerfield, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+and+In+Vitro+Fertilization+at+the+Zebrafish+International+Resource+Center.">Cryopreservation and In Vitro Fertilization at the Zebrafish International Resource Center.</a> Methods Mol. Biol. 546, 45-65 (2009).</li><li>Yang, H., Carmichael, C., Varga, Z. M., Tiersch, T. R. <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+a+simplified+and+standardized+protocol+with+potential+for+high-throughput+for+sperm+cryopreservation+in+zebrafish+Danio+rerio.">Development of a simplified and standardized protocol with potential for high-throughput for sperm cryopreservation in zebrafish Danio rerio.</a> Theriogenology. 68 (2), 128-136 (2007).</li><li>Morris, J. P., Berghmans, S., Zahrieh, D., Neuberg, D. S., Kanki, J. P., Look, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+sperm+cryopreservation+with+N,N-dimethylacetamide.">Zebrafish sperm cryopreservation with N,N-dimethylacetamide.</a> Biotechniques. 35 (5), 956-968 (2003).</li><li>Yoshizaki, 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=Spermatogonial+transplantation+in+fish:+A+novel+method+for+the+preservation+of+genetic+resources.">Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources.</a> Comp. Biochem. Physiol. Part D: Genomics Proteomics. 6 (1), 55-61 (2011).</li><li>Gao, D., Critser, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cryoinjury+in+living+cells.">Mechanisms of cryoinjury in living cells.</a> ILAR J. 41 (4), 187-196 (2000).</li><li>Asturiano, J. F., Cabrita, E., Horv&#225;th, &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress,+challenges+and+perspectives+on+fish+gamete+cryopreservation:+A+mini-review.">Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review.</a> Gen. Comp. Endocrinol. 245, 69-76 (2017).</li><li>K&#225;sa, 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=Development+of+sperm+vitrification+protocols+for+freshwater+fish+(Eurasian+perch,+Perca+fluviatilis)+and+marine+fish+(European+eel,+Anguilla+anguilla).">Development of sperm vitrification protocols for freshwater fish (Eurasian perch, Perca fluviatilis) and marine fish (European eel, Anguilla anguilla).</a> Gen. Comp. Endocrinol. 245, 102-107 (2017).</li><li>Cuevas-Uribe, R., Leibo, S. P., Daly, J., Tiersch, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+channel+catfish+with+sperm+cryopreserved+by+rapid+non-equilibrium+cooling.">Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling.</a> Cryobiology. 63 (3), 186-197 (2011).</li><li>Wang, Y., Xiao, Z., Li, L., Fan, W., Li, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+needle+immersed+vitrification:+A+practical+and+convenient+method+with+potential+advantages+in+mouse+and+human+ovarian+tissue+cryopreservation.">Novel needle immersed vitrification: A practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation.</a> Hum. Reprod. 23 (10), 2256-2265 (2008).</li><li>Liu, J., Cheng, K. M., Silversides, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Live+Offspring+from+Testicular+Tissue+Cryopreserved+by+Vitrification+Procedures+in+Japanese+Quail+(Coturnix+japonica).">Production of Live Offspring from Testicular Tissue Cryopreserved by Vitrification Procedures in Japanese Quail (Coturnix japonica).</a> Biol. Reprod. 88 (5), 124 (2013).</li><li>Liu, J., Cheng, K. M., Silversides, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+needle-in-straw+vitrification+can+effectively+preserve+the+follicle+morphology,+viability,+and+vascularization+of+ovarian+tissue+in+Japanese+quail+(Coturnix+japonica).">Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica).</a> Anim. Reprod. Sci. 134 (3-4), 197-202 (2012).</li><li>Lee, S., Yoshizaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+cryopreservation+of+spermatogonia+in+critically+endangered+Manchurian+trout+(Brachymystax+lenok).">Successful cryopreservation of spermatogonia in critically endangered Manchurian trout (Brachymystax lenok).</a> Cryobiology. 72 (2), 165-168 (2016).</li><li>Marques, L. 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=Viability+of+zebrafish+(Danio+rerio)+ovarian+follicles+after+vitrification+in+a+metal+container.">Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container.</a> Cryobiology. 71 (3), 367-373 (2015).</li><li>Luji&#263;, J., 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=First+successful+vitrification+of+salmonid+ovarian+tissue.">First successful vitrification of salmonid ovarian tissue.</a> Cryobiology. 76, 154-157 (2017).</li><li>Bono-Mestre, C., Cardona-Costa, J., Garc&#237;a-Xim&#233;nez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+on+cell+viability+of+three+zebrafish+testicular+cell+or+tissue+cryopreservation+methods.">Effects on cell viability of three zebrafish testicular cell or tissue cryopreservation methods.</a> CryoLetters. 30 (2), 148-152 (2009).</li><li>Marinovi&#263;, Z., 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=Cryosurvival+of+isolated+testicular+cells+and+testicular+tissue+of+tench+Tinca+tinca+and+goldfish+Carassius+auratus+following+slow-rate+freezing.">Cryosurvival of isolated testicular cells and testicular tissue of tench Tinca tinca and goldfish Carassius auratus following slow-rate freezing.</a> Gen. Comp. Endocrinol. 245, 77-83 (2017).</li><li>P&#353;eni&#269;ka, M., Saito, T., Rodina, M., Dzyuba, B. <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+early+stage+Siberian+sturgeon+Acipenser+baerii+germ+cells,+comparison+of+whole+tissue+and+dissociated+cells.">Cryopreservation of early stage Siberian sturgeon Acipenser baerii germ cells, comparison of whole tissue and dissociated cells.</a> Cryobiology. 72, 119-122 (2016).</li><li>Lee, S., Iwasaki, Y., Shikina, S., Yoshizaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+eggs+and+sperm+from+cryopreserved+whole+testes.">Generation of functional eggs and sperm from cryopreserved whole testes.</a> Proc. Nati. Acad. Sci. USA. 110 (5), 1640-1645 (2013).</li></ol>

Lo scopo principale di questo studio era di adattare l'ago immerso ultra-rapida procedura di raffreddamento sviluppato per specie di uccelli e mammiferi10,11,12 per la crioconservazione del testicolo di pesce (zebrafish come modello organismo). La maggior parte degli studi precedenti per quanto riguarda la crioconservazione delle risorse genetiche di zebrafish erano principalmente concentrata sulla crioconservazione di zebrafish...

Nuove tendenze nella scienza e nella biotecnologia hanno portato alla creazione di migliaia di nuove linee mutanti di topo, Drosophila, zebrafish e altre specie utilizzate come organismi modello in biomedica e altre scienze1. Inoltre, come nuove tecnologie vengono sviluppate e diventano disponibili, i numeri di linee mutanti costantemente aumentano2. Questo porta alla necessità per una conservazione sicura delle risorse genetiche oltre le comuni pratiche di mantenere colonie riproduttive. Come un metodo che consente l'archiviazione sicura delle risorse genetiche per un periodo di tempo indefinito, crioconservazione offre molti vantaggi come estensione della stagione riproduttiva, aggira la necessità per la manutenzione continua dei riproduttori, ed è più costo - e Labor-efficiente2.
Protocolli per la crioconservazione di spermatozoi sviluppato durante il passato parecchi anni2,3,4 offrono la possibilità di successo deposito di materiale genetico maschile di zebrafish. Tuttavia, la crioconservazione degli ovuli o embrioni di pesce non è ancora possibile grazie alla loro struttura complessa e di grandi quantità di materiale di tuorlo. Recentemente, la pratica del trapianto di cellule primordiali germinali (PGC) o cellule staminali spermatogoniali (SSCs) offre un bypass per questa barriera di svilupparsi dopo trapianto5in funzionali degli spermatozoi e uova. Crioconservazione delle CSS offre quindi una nuova frontiera nella conservazione delle risorse genetiche rare e preziose.
Anche se crioconservazione offre molti vantaggi, il processo di congelamento lento-tasso genera diverse condizioni che possono portare a cella danno2. Questi includono la formazione di ghiaccio intracellulare ed extracellulare, disidratazione, tossicità crioprotettore e altri. Ghiaccio intracellulare danneggia le cellule, ghiaccio extracellulare può portare a frantumazione meccanica delle cellule, mentre la diffusione dell'acqua dalle cellule durante il congelamento lento-tasso può condurre a disidratazione6. Recentemente, vetrificazione come una tecnica che impedisce gli effetti negativi della formazione di ghiaccio è stata applicata nella crioconservazione di gameti di pesce7,8,9. Esso presenta una tecnica di raffreddamento ultra-rapida attraverso il quale i mezzi di comunicazione interne ed esterne trasformare in uno stato amorfo/vetroso senza cristallizzazione in ghiaccio7,10. Vetrificazione successo del tessuto testicolare e ovarico è stata provata in specie di uccelli e mammiferi10,11,12, aprendo così la possibilità per la sua applicazione in pesce, pure.
In questo studio, presentiamo la procedura di vitrificazione (NIV) ago immerso per la crioconservazione di zebrafish tutta i testicoli. Dimostriamo un metodo affidabile per l'isolamento di cellule germinali di zebrafish fase iniziale senza contaminazione e un processo di crioconservazione che produce quantità elevate relativamente precoce delle cellule di germe con una bassa presenza di altre cellule, soprattutto gli spermatozoi. Al meglio della nostra conoscenza, questo è il primo studio a dimostrare un protocollo dettagliato visualizzato in quel momento per ultra-rapido raffreddamento del tessuto gonadico pesce e cellule germinali zebrafish. Inoltre, è evidenziato la ripetibilità del metodo in cinque differenti zebrafish ceppi: AB wild-type, casper (roy- / -; madreperla- / -), leopardo (leot1/t1), vasa [Tg (vas::eGFP)] e linea transgenica di Wilms tumore [Tg (wt1b::eGFP 1)].

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:232px;">Leibovitz media (L-15)</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:200px;">L1518</td>
			<td style="width:296px;">Supplemented with L-glutamine</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:232px;">Fetal bovine serum (FBS)</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:200px;">F9665</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tricaine methanesulfonate (MS-222)</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td>E10521</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:232px;">HEPES</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:200px;">H3375</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:232px;">Sucrose</td>
			<td style="width:220px;">Acros Organics</td>
			<td>57-50-1</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Trehalose</td>
			<td style="width:220px;">Acros Organics</td>
			<td>99-20-7</td>
			<td style="width:296px;">Dihydrate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Methanol</td>
			<td style="width:220px;">Reanal</td>
			<td style="width:200px;">20740-0-08-65</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Propylene glycol</td>
			<td style="width:220px;">Reanal</td>
			<td style="width:200px;">08860-1-08-65</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Dimethyl sulfoxide</td>
			<td style="width:220px;">Reanal</td>
			<td style="width:200px;">00190-1-01-65</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Collagenase</td>
			<td style="width:220px;">Gibco</td>
			<td style="width:200px;">9001-12-1</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Trypsin</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:200px;">T8003</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:232px;">DNase I</td>
			<td style="width:220px;">Panreac AppliChem</td>
			<td style="width:200px;">A3778</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Trypan blue</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:200px;">T6146</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Phospate buffered saline (PBS)</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td>P4417</td>
			<td>Tablets for preparation of 200 ml PBS solution</td>
		</tr>
	</tbody>
</table>

cryopreservation of zebrafish spermatogonia by whole testes needle immersed ultra-rapid cooling

Attuali tendenze nella scienza e nella biotecnologia portano alla creazione di migliaia di nuove linee in organismi modello, determinando così la necessità di nuovi metodi per la conservazione delle risorse genetiche oltre le comuni pratiche di mantenere colonie riproduttive. Lo scopo principale di questo studio era di adattare l'ago immerso procedura di vitrificazione (NIV) per crioconservare intero zebrafish testicoli. Crioconservazione delle cellule di germe iniziale di testicoli interi NIV offre possibilità per lo stoccaggio di zebrafish risorse genetiche, soprattutto perché dopo il trapianto può maturare in gameti maschili e femminili. Testicoli sono stati asportati, appuntato su un ago di agopuntura, equilibrato in due mezzi di Crioprotettivi (soluzione di equilibrazione contenente metanolo di 1,5 M e 1,5 M glicole propilenico; e soluzione di vitrificazione contenente dimetilsolfossido di 3 M e 3 M glicole propilenico) e ' immerso in azoto liquido. I campioni venivano riscaldati in una serie di tre soluzioni di riscaldamento conseguente. I principali vantaggi di questa tecnica sono (1) la mancanza di spermatozoi dopo digestione dei testicoli riscaldati, facilitando così le manipolazioni a valle; (2) ultra-rapido raffreddamento che permette l'esposizione ottima dei tessuti a azoto liquido quindi massimizzare il raffreddamento e ridurre la concentrazione necessaria di crioprotettori, riducendo la loro tossicità; (3) esposizione sincrono dei testicoli parecchie ai crioprotettori e azoto liquido; e ripetibilità (4) dimostra come ottenere redditività di oltre il 50% in cinque differenti zebrafish ceppi.

In media, il numero delle cellule di germe iniziale isolato da un testicolo singolo zebrafish fresco variava tra 40.000 e 200.000 cellule a seconda delle dimensioni del pesce. Quando nei testicoli di zebrafish fresco in tutti i 5 ceppi di digestione, cellule germinali primi non erano sola cellule presenti nelle sospensioni delle cellule (Figura 2). Al lato delle cellule di germe iniziale, numerosi gli spermatozoi sono stati trovati pure. D'altra parte, c'eran...

Watch this Scientific Journal Video about Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling at JoVE.com

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

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

Lo scopo principale di questo studio era di adattare l'ago immerso procedura di vitrificazione (NIV) per crioconservare intero zebrafish testicoli. Inoltre, è stata testata la ripetibilità del metodo in cinque zebrafish diversi ceppi.

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 ...

Cryopreservation offers the possibility of maintaining live cells and tissues at low temperatures for an infinite period of time, at least in human terms. Vitrification is a form of cryopreservation which involves ultra-rapid cooling into a glassy, amorphous state without the formation of crystalline ice and without the damages associated. In this video, we present the protocol for the vitrification of whole zebrafish testes, which allows their safe storage until their later use as a source of germ cells for transplantation into suitable recipients.The current vitrification protocol has one significant advantage over the slow-rate freezing protocols in regard to germ stem cell manipulations. And that is that after vitrification and warming, the spermatozoa die and they get adjusted so they are not present in the cell suspension. Therefore, there is no need for isolation of germ cells after warming.And that readily facilitates the downstream manipulations. Euthanize the fish by placing it into a dish containing 200 milligrams per liter of MS-222 at pH seven. Before continuing with the dissection, make sure that the gills have stopped moving, and at least 10 minutes have passed since that moment to ensure death by hypoxia.Dry the fish by patting it on a paper towel and place it on its dorsal side on a dissecting mat. Firstly, cut off the pelvic and pectoral fins for easier dissection. Horizontally snip the skin on the belly of the fish between the pectoral fins, and cut the skin and underlying muscle along the belly until the anal fin.Open the body cavity of the fish and pin the left and right body wall to the dissection mat with needles. Testes are located above the gastrointestinal organs on both sides of the swimming bladder. To prevent contamination, do not take out the gastrointestinal organs, but gently push them to one side and remove the testes from the opposing side.Testes are connected to the body wall, so remove them carefully and cut the connecting peritoneum by micro scissors. Additionally, in order to prevent contamination, firstly sterilize the testes by putting them into 70%ethanol for two seconds, and then place them in L-15 on 96-well plate on ice. Mark needles with appropriate labels, depending on the sample type, and prepare the equilibration and vitrification solutions.Transfer the testes to a larger-well plate or dish and pin three testes or more, depending on the size of the needle, on a sterile acupuncture needle. Firstly, position the testes on top of curved tweezers. Place the point of the needle in the middle of the testis and carefully pull the testis upward with the tweezers.After puncturing the testis, gently pull it upwards towards the top of the needle. Make sure the testes are separated from each other and they are not falling off or sliding down. Place the needles with pinned testes in a Tubulator Eppendorf tube filled with L-15 until vitrification at 25 degrees.The storage time should not exceed one hour. Transfer the needle with the testes into the equilibration solution and incubate for five minutes at 25 degrees. Transfer the needle into the vitrification solution and incubate for 30 seconds at 25 degrees.Remove the needle from the vitrification solution. Quickly and gently absorb the remaining solution from the tissue by a sterile paper towel. Use caution to avoid the testes sticking to the paper towel or falling off.Quickly place the needle into liquid nitrogen in a styrofoam box. Keep the needle in liquid nitrogen in a closed styrofoam box for five to 10 minutes. Pre-cool an opened 4.5 milliliter cryotube and its cap in the same box.After five to 10 minutes, transfer each needle into a separate cryotube under the surface of liquid nitrogen and close the cryotube. Place the cryotube onto a metal cane and place it in the cryobank canister as fast as possible. Store the samples in a canister storage drawer until further use.Warm each needle separately. All warming solutions should be at 25 degrees. Detach the cryotube from the metal cane and plunge it into liquid nitrogen stored in a styrofoam box.Open the cryotube in the liquid nitrogen vapor using forceps, and release the needle into the liquid nitrogen. Transfer the needle very fast into the first warming solution and incubate for one minute. Make sure to transfer the needle quickly to prevent it warming in the air during transfer.Transfer the needle into the second warming solution, and incubate for three minutes. Transfer the needle into the third warming solution and incubate for five minutes. Release the testes from the needle by sliding them downwards with curved tweezers.Place each warmed testis individually into a separate well filled with 250 microliters of L-15, supplemented with 10%FPS. Make sure that the wells are labeled according to the sample. Keep the well plate on ice until all samples are warmed, and until further work.Prepare 500 microliters of the dissociation solution for each sample in separate two-milliliter tubes. Label the tubes according to the code of the sample. Incubate the prepared dissociation solution for five minutes at room temperature.Add the warmed tissue into the digestion solution, and cut it into small pieces by at least 30 movements of small scissors. Incubate the tissue on a shaking plate for 90 minutes at 24 degrees. Stop the digestion process by adding 400 microliters of L-15 and 100 microliters of 10%FPS.Briefly shake the solution and incubate for one minute at room temperature. Filter the obtained solution through 50-micron filters into a 1.5 milliliter tube. Label the tubes accordingly.Centrifuge the filtered solution at 200 G for 10 minutes at 25 degrees. Carefully remove the supernatant and re-suspend the pellet in 20 microliters of L-15, supplemented with 10%FPS. Manually shake the tube until the pellet is completely dissolved.Keep the cell suspension on ice or at four degrees until further use. Mix five microliters of cell suspension and five microliters of 0.4%trypan blue solution in an appropriately-labeled 0.2 milliliter PCR tube. If different volumes are used, make sure that the dilution ratio of one to one remains.Incubate this suspension for one to three minutes at 25 degrees. Check the viability of each sample in a hemocytometer under a microscope. Count the number of live cells unstained by trypan blue in 15 fields.Calculate the total number of cells in 20 microliters of the cell suspension. The suspension is now ready for any downstream application. On average, the number of early-stage germ cells isolated from a single fresh zebrafish testis vary between 40, 000 and 200, 000 cells, depending on the size of the fish.In all zebrafish lines used in this study, the current vitrification protocol yielded average viability rates of 50%and higher. This indicates favorable viability and repeatability of the presented protocol. When digesting fresh zebrafish testes in all five strains, besides the early-stage germ cells, numerous spermatozoa were found as well.After digestion of cryo-preserved testes, there were far less spermatozoa, indicating that they do not survive this vitrification protocol, and that they are most likely eliminated during the digestion process. The needle-immersed vitrification protocol presented in this study offers several advantages in the cryopreservation of testicular pieces. Firstly, several pieces can be pinned to one needle, and therefore they can simultaneously be exposed to cryoprotectants and to the liquid nitrogen.Furthermore, this method enables that very low amounts of cryoprotectants are attached to the tissue pieces. And therefore, cooling and warming rates can be higher. One significant advantage is that spermatozoa do not survive this protocol, and therefore in the end, the cell suspensions are enriched spermatogonia, which will facilitate the downstream applications.But one thing that has to be taken into account is the possibility of cross-contamination through nitrogen. Therefore when using this method, liquid nitrogen should not be reused.

Research

JoVE Journal

Biology

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Il trapianto di midollo renale intero in Zebrafish adulti

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Ricerca

Biologia

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish intero monte ad alta risoluzione doppia fluorescente In Situ Ibridazione

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Generating Chimeric Zebrafish Embryos by Transplantation

Generare embrioni di zebrafish Chimerico da trapianto

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

A High-Throughput Metodo per la crioconservazione dello sperma e Zebrafish In Vitro Fecondazione

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Making Gynogenetic Diploid Zebrafish by Early Pressure

Fare Diploide Zebrafish Gynogenetic di Pressure presto

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Isolation of Drosophila melanogaster Testes

Isolation of Drosophila melanogaster Testes

Isolamento di Drosophila melanogaster Testicoli

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Crioconservazione di embrioni di topo da glicole etilenico-Based Vetrificazione

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

Imaging Centrosomes in Fly Testes

I centrosomi Imaging in Fly Testicoli

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Analisi di apoptosi in embrioni di zebrafish da Whole-mount immunofluorescenza per rilevare caspasi 3 attivata

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

Preparazione di montaggio schermo per l&#39;osservazione e l&#39;analisi di zebrafish campioni macchiato da Whole Monte<em&gt; In situ</em&gt; Ibridazione

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...