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

This study was supported by the National Research, Development and Innovation Office of Hungary (grant 116912 to &#193;H), the COST office (Food and Agriculture COST Action FA1205: AQUAGAMETE), the Stipendium Hungaricum Scholarship Programme (grant to ZM) and the New Hungarian National Excellence Predoctoral Fellowship (grant to EK).

All methods described here have been approved by the Hungarian Animal Welfare Law.
1. Reagent Preparation
<ol>
	<li>Stock solutions
	<ol>
		<li>Prepare 1 M trehalose by adding 0.378 g of trehalose dihydrate into 1 mL of dH2O. Mix well until it dissolves completely.</li>
		<li>Prepare 1 M sucrose by adding 0.342 g of sucrose into 1 mL of dH2O. Mix well until it dissolves completely.</li>
		<li>Prepare 1 M HEPES by&#160;adding 0.238 g of HEPES to 1 mL of dH2O. ...

<ol>
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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>

The main purpose of this study was to adapt the needle immersed ultra-rapid cooling procedure developed for avian and mammalian species10,11,12 to the cryopreservation of fish testis (zebrafish as a model organism). Most of the previous studies regarding cryopreservation of zebrafish genetic resources were primarily focused on cryopreservation of zebrafish sperm2,3,...

Novel trends in science and biotechnology have led to the creation of thousands of new mutant lines of mice, Drosophila, zebrafish and other species used as model organisms in biomedical and other sciences1. Furthermore, as new technologies are developed and become available, the numbers of mutant lines steadily increase2. This leads to the necessity for a safe storage of genetic resources beyond the common practices of keeping breeding colonies. As a method which enables safe storage of genetic resources for an indefinite period of time, cryopreservation offers many advantages such as extension of reproductive season, circumvents the need for continuous maintenance of broodstock, and it is more cost- and labor-efficient2.
Protocols for sperm cryopreservation developed during the past several years2,3,4 offer the opportunity for successful storage of zebrafish male genetic material. However, cryopreservation of eggs or embryos in fish is not yet possible due to their complex structure and large amounts of yolk material. Recently, the practice of transplantation of primordial germ cells (PGCs) or spermatogonial stem cells (SSCs) offers a bypass to this barrier by developing into functional sperm and eggs after transplantation5. Therefore, cryopreservation of SSCs offers a new frontier in conservation of rare and valuable genetic resources.
Even though cryopreservation offers many advantages, the slow-rate freezing process generates several conditions that may lead to cell damage2. These include intracellular and extracellular ice formation, dehydration, cryoprotectant toxicity and others. Intracellular ice damages the cells, extracellular ice may lead to mechanical crushing of cells, while water diffusion from the cells during slow-rate freezing may lead to dehydration6. Recently, vitrification as a technique which prevents the negative effects of ice formation has been applied in the cryopreservation of fish gametes7,8,9. It presents an ultra-rapid cooling technique through which the internal and external media turn into an amorphous/glassy state without crystalizing into ice7,10. Successful vitrification of testicular and ovarian tissue has been evidenced in avian and mammalian species10,11,12, thus opening possibilities for its application in fish, as well.
In this study, we present the needle immersed vitrification (NIV) procedure for the cryopreservation of whole zebrafish testes. We demonstrate a reliable method for the isolation of zebrafish early-stage germ cells without contamination and a cryopreservation process that yields relatively high amounts of early-stage germ cells with a low presence of other cells, especially spermatozoa. To the best of our knowledge, this is the first study to demonstrate a detailed visualized protocol for ultra-rapid cooling of fish gonadal tissue and zebrafish germline cells. Additionally, repeatability of the method is demonstrated in five different zebrafish strains: AB wild type, casper (roy-/-; nacre-/-), leopard (leot1/t1), vasa [Tg(vas::eGFP)] and Wilms tumor [Tg(wt1b::eGFP 1)] transgenic line.

<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

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

On average, the number of early-stage germ cells isolated from a single fresh zebrafish testis varied between 40,000 and 200,000 cells depending on the size of the fish. When digesting fresh zebrafish testes in all 5 strains, early-germ cells were not the only cells present in the cell suspensions (Figure 2). Beside the early-stage germ cells, numerous spermatozoa were found as well. On the other hand, there were far less spermatozoa after digestion of cryopr...

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

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

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

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