Name: in vitro culture strategy for oocytes from early antral follicle in cattle
Uploaded: 2020-07-08T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle at JoVE.com

这项工作得到了伦巴第地区PSR INNOVA n.201801061529和联伊特派团n.PSR 2019_DIP_027_ALUCI_01

在当地屠宰场（INALCA S.p.A.，奥斯佩达莱托·洛迪迪亚诺，LO，IT 2270M CE，意大利）回收的4至8岁的荷尔斯泰因奶牛。
1. 媒体准备
注：所有介质在使用前必须至少准备四小时。碳酸氢钠缓冲介质在38.5°C和5%CO2空气中孵育，最大湿度。2HEPES 缓冲介质在恒温烤箱中保持在 38.5 °C。
<ol><li>卵母细胞（L-IVCO）介质的长体外培养<ol>
<li>准备15 mL的基本培养基?...

<ol>
	<li>Lonergan, P., Fair, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+Oocytes+in+Vitro.">Maturation of Oocytes in Vitro.</a> Annual Review of Animal Biosciences. 4, 255-268 (2016).</li><li>Luciano, A. M., Franciosi, F., Modina, S. C., Lodde, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junction-mediated+communications+regulate+chromatin+remodeling+during+bovine+oocyte+growth+and+differentiation+through+cAMP-dependent+mechanism(s).">Gap junction-mediated communications regulate chromatin remodeling during bovine oocyte growth and differentiation through cAMP-dependent mechanism(s).</a> Biology of Reproduction. 85 (6), 1252-1259 (2011).</li><li>McLaughlin, M., Telfer, E. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+in+vitro+maturation+of+oocytes:+a+matter+of+follicular+differentiation.">Successful in vitro maturation of oocytes: a matter of follicular differentiation.</a> Biology of Reproduction. 98 (2), 162-169 (2018).</li><li>Lonergan, P., Fair, T. <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-produced+bovine+embryos:+dealing+with+the+warts.">In vitro-produced bovine embryos: dealing with the warts.</a> Theriogenology. 69 (1), 17-22 (2008).</li><li>Clement, M. D. F., Dalbies-Tran, R., Estienne, A., Fabre, S., Mansanet, C., Monget, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ovarian+reserve+of+primordial+follicles+and+the+dynamic+reserve+of+antral+growing+follicles:+what+is+the+link.">The ovarian reserve of primordial follicles and the dynamic reserve of antral growing follicles: what is the link.</a> Biology of Reproduction. 90 (4), 85 (2014).</li><li>Lussier, J. G., Matton, P., Dufour, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+rates+of+follicles+in+the+ovary+of+the+cow.">Growth rates of follicles in the ovary of the cow.</a> Journal of Reproduction and Fertility. 81 (2), 301-307 (1987).</li><li>Fair, T., Hulshof, S. C., Hyttel, P., Greve, T., Boland, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+ultrastructure+in+bovine+primordial+to+early+tertiary+follicles.">Oocyte ultrastructure in bovine primordial to early tertiary follicles.</a> Anatomy and Embryology (Berlin). 195 (4), 327-336 (1997).</li><li>Pavlok, A., Lucas-Hahn, A., Niemann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+and+developmental+competence+of+bovine+oocytes+derived+from+different+categories+of+antral+follicles.">Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles.</a> Molecular Reproduction and Development. 31 (1), 63-67 (1992).</li><li>Blondin, P., Sirard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+and+follicular+morphology+as+determining+characteristics+for+developmental+competence+in+bovine+oocytes.">Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes.</a> Molecular Reproduction and Development. 41 (1), 54-62 (1995).</li><li>Harada, 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=Bovine+oocytes+from+early+antral+follicles+grow+to+meiotic+competence+in+vitro:+effect+of+FSH+and+hypoxanthine.">Bovine oocytes from early antral follicles grow to meiotic competence in vitro: effect of FSH and hypoxanthine.</a> Theriogenology. 48 (5), 743-755 (1997).</li><li>Hirao, Y., 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+growth+and+development+of+bovine+oocyte-granulosa+cell+complexes+on+the+flat+substratum:+effects+of+high+polyvinylpyrrolidone+concentration+in+culture+medium.">In vitro growth and development of bovine oocyte-granulosa cell complexes on the flat substratum: effects of high polyvinylpyrrolidone concentration in culture medium.</a> Biology of Reproduction. 70 (1), 83-91 (2004).</li><li>Alm, H., Katska-Ksiazkiewicz, L., Rynska, B., Tuchscherer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+meiotic+competence+of+bovine+oocytes+originating+from+early+antral+ovarian+follicles.">Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles.</a> Theriogenology. 65 (7), 1422-1434 (2006).</li><li>Taketsuru, H., 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=Bovine+oocytes+in+secondary+follicles+grow+in+medium+containing+bovine+plasma+after+vitrification.">Bovine oocytes in secondary follicles grow in medium containing bovine plasma after vitrification.</a> Journal of Reproduction and Development. 57 (1), 99-106 (2011).</li><li>Endo, 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=Estradiol+supports+in+vitro+development+of+bovine+early+antral+follicles.">Estradiol supports in vitro development of bovine early antral follicles.</a> Reproduction. 145 (1), 85-96 (2013).</li><li>Makita, M., Miyano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+hormones+promote+bovine+oocyte+growth+and+connection+with+granulosa+cells.">Steroid hormones promote bovine oocyte growth and connection with granulosa cells.</a> Theriogenology. 82 (4), 605-612 (2014).</li><li>Yamamoto, K., 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+to+live+young+from+bovine+small+oocytes+after+growth,+maturation+and+fertilization+in+vitro.">Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro.</a> Theriogenology. 52 (1), 81-89 (1999).</li><li>Alam, M. 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C. 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=Steroid+hormones+interact+with+natriuretic+peptide+C+to+delay+nuclear+maturation,+to+maintain+oocyte-cumulus+communication+and+to+improve+the+quality+of+in+vitro-produced+embryos+in+cattle.">Steroid hormones interact with natriuretic peptide C to delay nuclear maturation, to maintain oocyte-cumulus communication and to improve the quality of in vitro-produced embryos in cattle.</a> Reproduction, Fertililty and Development. 29 (11), 2217-2224 (2017).</li><li>Soares, A. C. 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=Characterization+and+control+of+oocyte+large-scale+chromatin+configuration+in+different+cattle+breeds.">Characterization and control of oocyte large-scale chromatin configuration in different cattle breeds.</a> Theriogenology. 141, 146-152 (2020).</li><li>Franciosi, 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=Natriuretic+peptide+precursor+C+delays+meiotic+resumption+and+sustains+gap+junction-mediated+communication+in+bovine+cumulus-enclosed+oocytes.">Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes.</a> Biology of Reproduction. 91 (3), 61 (2014).</li><li>Luciano, A. 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在这里，我们描述了一个培养卵母细胞的培养系统，通过支持卵母细胞的生存能力和防止卵母细胞的恢复，促进卵母细胞发育5天。后一个方面是最重要的，让持续生长和分化是必要的，使卵母细胞与小鼠和胚胎发育能力2,2，20，否则将阻止过早恢复的美的分裂。
在开发这种培养系统时，我们考虑了卵泡中生理生长和分化的几个特征...

在生殖寿命期间，卵巢成熟中存在的卵母细胞中，只有极小部分在排卵管中排卵，并可用于受精和发育成可行的胚胎1。另一方面，卵巢内大多数卵母细胞都经过卵巢，从不排卵。体外胚胎生产（IVP）技术试图增加卵巢储备2，3,的利用。迄今为止，这些技术允许对生育保护、牲畜选择方案以及保护濒危物种进行干预。然而，大多数协议使用已基本完成在蚁卵巢卵泡内生长阶段的卵母细胞，因此被称为完全生长的卵母细胞。在广泛采用 IVP 技术的牛中，完全生长的卵母细胞的最终直径约为 120 μm，从直径为 2 至 8 mm 的卵泡（中等蚂蚁卵泡）1 中采集。与卵泡分离后，这种卵母细胞在体外成熟和受精。然后，酶被培养到囊肿阶段，或转移到接受者或冷冻保存。在牛和许多其他物种中，尽管 IVP 提供了潜力，但每头牛的体外胚胎数量过去 40 年并没有大有改善。这部分是由于在给定时间填充卵巢的完全生长的卵母细胞数量有限，可以检索并接受标准 IVP 技术4、,5、6。
封闭在早期蚁卵泡内的卵母细胞，即直径小于2毫米的卵泡，是生育保全计划7中的潜在来源，因为卵巢中大约含有比中蚂蚁8多10倍的早期蚁卵泡。然而，这些卵母细胞仍处于生长阶段，尚未达到完全生长阶段9。因此，他们仍然具有转录活性，产生mRNA，将储存为以后的发展步骤，尚未经历所需的所有分化过程，赋予卵母细胞的能力，自发地恢复和完成美病我曾经从卵泡舱10，11,分离。因此，它们不能直接提交到标准体外成熟 （IVM） 协议，但它们需要额外的培养期，以便它们能够完成生长阶段并正确区分。
从生长阶段到完全生长阶段的过渡，在牛中，当毛囊从早期的蚁体发展到中蚁体阶段时，是卵母细胞发育的关键步骤之一。在牛中，多项研究试图在体,外,,2、12、13、14、15、16、17、18、192,12,13,1416,1718中重新概括这些事件。15然而，迄今尚未制定可靠的议定书，只报告了有限的成功。根据先前的研究20，这些生长的卵母细胞构成一个同质种群。除了转录活性外，它们的染色质还分散在细菌囊泡（GV）中，其配置名为GV02，21。,21相反，从中等蚂蚁卵泡中获得的完全生长的卵母细胞的种群是更异质的，这种条件由不同程度的染色质压实（GV1、GV2和GV3）所反映，可以观察到20。其中，此前的数据表明，GV2和GV3卵母细胞总体具有较好的品质和较高的胚胎发育能力20、21、22、23、24。20,21,22,23,24
从上述观察开始，我们在此描述了一个5天长的卵母细胞培养系统（L-IVCO），它允许从早期的蚁卵泡中分离出分离为积细胞-卵母细胞复合物（COCs）的卵母细胞。这种培养策略是从我们实验室进行的10年长期研究演变而来的，其基础是先前开发的体外卵母细胞培养（IVCO）2，早产系统23，25,25和卵母细胞培养期间的锌补充。卵泡刺激激素（FSH）和磷酸二酯酶-3（PDE3）抑制剂的组合，能够增强积糖-卵母细胞传播2，防止不合时宜的美异恢复2，并支持卵母细胞生长2被使用。

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			<td height="20" style="height:21px;width:215px;">4-well dishes</td>
			<td style="width:127px;">Nunclon</td>
			<td style="width:123px;">179830</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:215px;">96-well dish</td>
			<td style="width:127px;">Becton Dickinson Biosciences</td>
			<td style="width:123px;">356649</td>
			<td>BioCoat&#8482; Collagen I</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:215px;">Bovine Serum Albumin (Fatty acid free)</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">A8806</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Bovine Serum Albumin (Fraction V)</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">A3311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Cell culture water</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">W3500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Cilostamide</td>
			<td style="width:127px;">Sigma</td>
			<td>C7971</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Cysteamine</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">M9768</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Digital camera</td>
			<td style="width:127px;">Nikon Corp</td>
			<td>Camera DS-5M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Disodium phosphate</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">S5136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Estradiol</td>
			<td>Sigma</td>
			<td>E2758</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Glutamax Supplement</td>
			<td style="width:127px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">35050061</td>
			<td></td>
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			<td height="21" style="height:21px;width:215px;">Gonal F</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Heparin</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">H3149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Hepes</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">H3784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Vacuum pump</td>
			<td>Cook-IVF</td>
			<td style="width:123px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:215px;">Incubator</td>
			<td style="width:127px;">Sanyo</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Kanamycin sulfate from Streptomyces kanamyceticus</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">K1377</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:45px;width:215px;">Medium 199</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">M3769</td>
			<td style="width:171px;">Powder for hepes-buffered TCM199</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;width:215px;">Medium 199</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">M2520</td>
			<td>Powder for M199-D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Microscope</td>
			<td>Nikon Corp</td>
			<td>Nikon Diaphot</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Microscope</td>
			<td style="width:127px;">Nikon Corp</td>
			<td style="width:123px;">Eclipse E 600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Monopotassium phosphate</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P5655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">158127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Penicilin</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P3032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Phenol Red</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P5530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Polyvinyl alcohol</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P8137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Polyvinylpyrrolidone</td>
			<td style="width:127px;">Sigma</td>
			<td>P5288</td>
			<td>360k molecular weight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Potassium chloride</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P5405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Progesterone</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P8783</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium bicarbonate</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium choride</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P5886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium pyruvate</td>
			<td style="width:127px;">Sigma</td>
			<td>P4562</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Streptomycin</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">S9137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Testosterone</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">86500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Triton X</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">T9284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Vectashield with DAPI</td>
			<td style="width:127px;">Vector Laboratories</td>
			<td style="width:123px;">H1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water</td>
			<td>Sigma</td>
			<td style="width:123px;">W3500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Zinc sulfate heptahydrate</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">Z0251</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

成熟、可受精的卵母细胞的有限储备是哺乳动物辅助生殖成功的主要障碍。考虑到在生殖寿命期间，卵巢成熟和排卵中只有约1%的卵母细胞，已开发出若干技术，以增加卵巢储备对越来越多的非卵巢卵泡种群的利用。这些技术允许对生育保护、牲畜选择方案以及濒危物种的保护进行干预。然而，卵巢储备的巨大潜力在很大程度上仍未开发。例如，在奶牛中，已经尝试在特定的发育阶段支持卵母细胞的体外培养，但尚未制定有效和可靠的方案。在这里，我们描述了一个培养系统，它再现了相应卵泡阶段的生理条件，被定义为从牛早期蚁卵泡中收集到完全生长的卵母细胞，与体内的中蚂蚁卵泡相对应。激素和磷酸二酯酶3抑制剂的组合被用来防止不合时宜的美的恢复，并指导卵母细胞的分化。

在L-IVCO结束时，COC的形态变化，根据积液细胞的外观确定了4个类，如图2所示。根据选择健康COCs11、26、27的通常形态标准，1、2和3类被判定为健康，而4类则表现出明显的退化迹象，如卵母细胞周围没有完整的积细胞层，被认为严重受损，不适合在未来的 IVP 设置中接受下游程序。11,26,27总的来说，对5个生...

Watch this Scientific Journal Video about In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle at JoVE.com

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

我们描述了在发育的早期阶段从卵巢卵泡分离生长的卵母细胞的过程，以及建立体外培养系统，该系统可以支持生长和分化，一次，一次，一次，一个发育阶段。

牛早期蚁卵泡卵母细胞的体外培养策略

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

在此视频协议中，我们详细演示了一种培养牛卵母细胞的培养系统。该系统支持卵母细胞在培养中的可行性长达五天，并允许其分化。利用这种培养系统，可以生长从小蚂蚁卵泡中收集的卵母细胞，这些卵母细胞通常不用于生产体外的标准协议中，以增加可受精配子的可用性。这种对更广泛保护区的开采，可以为受威胁物种、牛家族或面临遗传侵蚀风险的当地品种的遗传抢救提供新的选择。首先，准备15毫升的基本培养本，并按照手稿指示进行补充。接下来，准备三毫升的保持介质，添加五微摩尔西洛酰胺到基本培养基，并倒入35毫米培养皿。根据手稿方向补充基本文化媒介，准备长IVCO介质。在 96 层涂层板的每个井中加入 200 微升长 IVCO 介质。然后用无菌水填充四个边缘，以补偿蒸发，并在培养过程中保持适当的湿度。在38.5摄氏度和5%的二氧化碳下孵育96井板和保持介质，湿度最大。在无菌盐水中清洗卵巢四次，保持在26摄氏度。然后吸气所有直径超过两毫米的毛囊，用18个量表的针连接到吸气泵。将吸气卵巢放在烧杯中，无菌盐水保持在26摄氏度。一次将一个卵巢放在无菌PTFE切割板上，并使用手术号22刀片切割1.5至2毫米厚的卵巢皮层，并平行于器官的主要轴。将卵巢皮层片放在无菌玻璃培养皿中，在38.5摄氏度的温盘中用解剖介质。在60毫米玻璃培养皿中，用2到3毫升的M199D在解剖显微镜下，选择5至2毫米之间的卵泡。通过观察形态参数（如均匀明亮的半透明外观、广泛的血管化和常规的格兰努洛萨层）在立体显微镜下识别健康的非反应性毛囊。虽然阁楼毛囊具有灰色不透明的外观，并且血管化不良，内部有深色 COC。丢弃阿克雷特毛囊，处理所有其他。使用手术刀片去除一侧毛囊周围的卵巢组织，直到其暴露。然后使用 26 量表针小心地在暴露的卵泡壁中进行一个缝隙，从而释放包括 COC、卵泡液和细胞团块的卵泡含量。识别 COC 并检查其积原完整性、zona pellucida 完整性和细胞质的同质性。如果符合这些标准，则使用 P20 移液器吸气 COC。将隔离的 COC 与 M199D 西罗斯塔米德进行分离，然后继续隔离程序 30 分钟。选择COC后，如前所述，在60毫米培养皿中用20微升的M199D西洛斯塔米德准备16滴，并在每滴中放入一个健康的COC。使用连接到相机的倒置显微镜测量卵母细胞直径，不包括佐纳佩普西达。通过清晰的卵母细胞可视化，进行两次垂直测量，确保两次测量的平均值在 100 到 110 微米的范围内。由于伴生积细胞，很难精确测量卵母细胞直径。具有直径较小或较大的卵母细胞，或没有圆形卵母细胞的COC，或具有无法测量的卵母细胞的COC应丢弃。将选定的 COC 转移到含有 M199H 介质的 35 毫米培养皿中，并在 38.5 摄氏度和 5%的二氧化碳中保持最大湿度，确保总工作时间不超过两小时。一旦选择和收集程序完成，将一个COC转移到以前准备的96井板的每个井的中心。在38.5摄氏度和5%的二氧化碳最大湿度下孵育5天，每隔一天刷新一次。要更新介质，请用 100 微升的新鲜长 IVCO 介质更换 100 微升旧介质，请使用立体显微镜进行更换，以避免移动 COC。在长IVCO结束时，分析COC形态。如果他们有一个紧凑的积子细胞投资没有积血扩张的迹象，或细胞退化，将它们分类为一类。如果 COC 具有紧凑的积子细胞投资，没有积率膨胀或细胞退化的迹象，并且具有一个或多个类状的形成，则将它们分类为第二类。第三类COC，显示几层积细胞，没有积血扩张的迹象，一些分类细胞在外层积血细胞和没有蚂蚁样的形成。如果COCs显示大量积细胞损失超过卵母细胞表面的50%，以及细胞退化和细胞碎片的迹象，则将其归类为第四类。在长IVCO结束时，COC的形态发生了变化。根据积细胞的外观确定了四个类。总的来说，对5个生物复制体中的74个卵母细胞进行了分析，其中9.45%在进一步评估中被丢弃。第一类、第二类和三类被判定为健康，而第四类则表现出明显的退化迹象，例如卵母细胞周围没有完整的积细胞层。这些 COC 被认为不适合在未来的 IVP 环境中进行下游程序。在长IVCO结束时对卵母细胞的评估表明，在未成熟阶段，仍有更高比例的卵母细胞被逮捕，而染色质仍被封闭在GV内。当GV内的染色质凝结被调查为能力提升的标志时，在59%的卵母细胞中观察到染色质配置向更浓缩的阶段（即GV 2和GV 3）的过渡。一小部分恢复性，达到元相一个阶段，或退化。这些结果表明，L-IVCO培养支持卵母细胞的生存能力，同时防止五天期复生。在长IVCO结束时，COC可用于体外胚胎生产的下游程序，即体外成熟、受精和胚胎培养。这些步骤是必要的，以提供概念证明，卵母细胞已经获得了更高的发展能力。除了拥有增加受精卵母细胞数量的潜力外，长IVCO培养系统对于有兴趣解剖调节能力游戏体形成的细胞和分子过程的科学家来说，也是一种工具。

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JoVE Journal

Developmental Biology

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

了解早期器官使用简体<em&gt;原位</em&gt;杂交协议<em&gt;爪</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

科研

发育生物学

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

基于核仁染色质组织分离鼠标窦卵母细胞与表征

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

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

人多能干细胞对原始和最终造血祖细胞定向分化的研究

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

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

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

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

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

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

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

人类羊膜不同解剖区域的皮下细胞体外培养

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...