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

这项研究得到了匈牙利国家研究、发展和创新办公室 (赠款116912至ÁH)、成本办公室 (粮食和农业成本行动 FA1205: AQUAGAMETE)、Stipendium Hungaricum 奖学金方案 (赠款给各部) 和新匈牙利国家优秀 Predoctoral 奖学金 (授予克朗)。

这里描述的所有方法都已获得匈牙利动物福利法的批准。
1. 试剂制备
<ol><li>库存解决方案<ol>
<li>将1米海藻糖加入0.378 克海藻糖, 放入1毫升的 dH2o 混合均匀, 直到完全溶解。</li>
		<li>将1米蔗糖加入0.342 克蔗糖, 放入1毫升的 dH 中2o 混合均匀, 直至完全溶解。</li>
		<li>1米 HEPES 通过添加0.238 克 HEPES 到1毫升的 dH2o 混合好, 直到它完全溶解。</li>
		<li>通过增?...

<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>

本研究的主要目的是适应为禽类和哺乳动物物种开发的针浸入式超快速冷却程序10,11,12冷冻鱼睾丸 (斑马鱼作为模型有机体)。以往关于斑马鱼遗传资源超低温保存的研究主要集中在斑马鱼精子的超低温保存上2,3,4。然而, 成熟卵母细胞和胚胎的冷冻保存协议?...

科学和生物技术方面的新趋势导致创造了数以千计的新的突变体, 如小鼠、果蝇、斑马鱼和其他物种, 它们被用作生物医学和其他科学中的模范有机体1。此外, 随着新技术的开发和使用, 变异线的数量稳步增加2。这就导致了在保持繁殖殖民地的共同做法之外, 安全储存遗传资源的必要性。冷冻保存作为一种保证遗传资源在不确定时间内安全贮存的方法, 具有延长繁殖季节的优势, 绕过了对亲鱼持续维护的需要, 成本更高, 而且劳动效率2。
在过去几年中开发的精子超低温保存协议2,3,4为斑马鱼雄性遗传物质的成功储存提供了机会。然而, 由于其结构复杂, 蛋黄物质大量存在, 鱼的卵或胚胎的超低温保存还是不可能的。最近, 移植的原始生殖细胞 (PGCs) 或精原干细胞 (干细胞) 提供了一个旁路到这个障碍通过发展成功能精子和卵子移植后的5。因此, 干细胞的超低温保存为珍稀珍贵的遗传资源的保护提供了新的前沿。
尽管超低温保存提供了许多优点, 但慢速冻结过程会产生几种可能导致细胞损伤的条件2。其中包括细胞内和胞外冰的形成、脱水、cryoprotectant 毒性等。细胞内的冰会损伤癌细胞, 细胞外的冰可能会导致机械破碎, 而在慢速冷冻过程中细胞的水扩散可能导致脱水6。最近, 玻璃化作为一种防止冰形成的负面影响的技术已被应用于鱼类配子的超低温保存7,8,9。它提供了一种超快速冷却技术, 通过它, 内部和外部介质变成非晶态/玻型状态而不 crystalizing 到冰7,10。成功的玻璃化睾丸和卵巢组织已证明在鸟类和哺乳动物物种10,11,12, 从而打开它的应用在鱼的可能性, 以及。
在本研究中, 我们提出了针浸泡玻璃化的程序, 以保存整个斑马鱼睾丸。我们展示了一种可靠的方法, 以隔离斑马鱼的早期生殖细胞没有污染和冷冻过程, 产生相对较高数量的早期生殖细胞与低存在的其他细胞, 特别是精子。根据我们的知识, 这是第一项研究, 以演示一个详细的可视化协议, 以超快速冷却鱼类性腺组织和斑马鱼生殖细胞。此外, 该方法的重复性表现在五种不同的斑马鱼菌株: AB 野生型, 精灵 (罗伊-/;珍珠层-/), 豹 (leot1/t1), [tg (输精管:: eGFP)] 和肾母细胞肿瘤 [tg (wt1b::eGFP1)] 转基因线。

<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

目前科学和生物技术的趋势导致在模型生物体中创造数以千计的新品系, 从而导致有必要采用新的方法来安全地储存遗传资源, 使之超越饲养殖民地的共同做法。本研究的主要目的是适应针浸泡玻璃化 (cryopreserve) 程序, 以使整个斑马鱼睾丸。早期生殖细胞的全睾丸冷冻保存为斑马鱼遗传资源的贮藏提供了可能, 特别是在移植后它们可以成熟为雄性和雌性配子。睾丸被切除, 固定在针刺针上, 平衡在两个低温保护介质中 (含1.5 米甲醇和1.5 米丙烯乙二醇的平衡溶液; 含有3米二甲基亚砜和3米丙二醇的玻璃化溶液)并陷入液态氮。在一系列的三种升温溶液中, 样品得到了升温。这项技术的主要优点是 (1) 在热睾丸消化后缺乏精子, 从而促进下游操作;(2) 超快速冷却, 使组织的最佳接触液氮, 从而最大限度地冷却和减少所需的冷冻保护剂浓度, 从而减少其毒性;(3) 多睾丸同步暴露于冷冻保护剂和液氮;(4) 可重复性表现为在五种不同的斑马鱼菌株中获得50% 以上的生存能力。

平均来说, 从一个新鲜的斑马鱼睾丸中分离出来的早期生殖细胞的数量在4万到20万个细胞之间变化, 这取决于鱼的大小。在所有5株中消化新鲜的斑马鱼睾丸时, 早期生殖细胞并不是细胞悬浮液中唯一存在的细胞 (图 2)。在早期生殖细胞旁边, 也发现了许多精子。另一方面, 在冷冻后的睾丸中, 精子的消化少得多。这表明精子在超快速冷却协议中无法存活,...

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

全睾丸针浸泡超快速冷却法超低温保存斑马鱼细胞

本研究的主要目的是适应针浸泡玻璃化 (cryopreserve) 程序, 以使整个斑马鱼睾丸。另外, 对五种不同斑马鱼菌株的重复性进行了测试。

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

在成年斑马鱼的全肾骨髓移植

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

科研

生物学

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

斑马鱼整装高分辨率双荧光原位杂交

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

移植嵌合斑马鱼胚胎生成

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

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

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

初压雌核发育二倍体的斑马鱼

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

分离果蝇睾丸

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

基于乙二醇玻璃化冷冻小鼠胚胎的超低温保存

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

在飞睾丸成像中心体

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

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

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

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