Summary

从绵羊的成人和前卵巢收集的体外成熟卵母细胞的振动

Published: July 10, 2021
doi:

Summary

该议定书旨在为成年和幼羊卵母细胞的玻璃化提供标准方法。它包括从体外成熟介质的准备到暖化后文化的所有步骤。卵母细胞在 MII 阶段使用 Cryotop 进行振动,以确保最低基本体积。

Abstract

在牲畜中,体外胚胎生产系统可以开发和维持,这要归功于大量卵巢和卵母细胞,这些卵巢和卵母细胞很容易从屠宰场获得。成人卵巢总是携带几个心脊囊,而在青春期前的捐赠者中,卵母细胞的最大数量在4周大时可用,而卵巢承担蚂蚁卵泡的峰值数量。因此,4周大的羔羊被认为是很好的捐赠者,即使与成年卵母细胞相比,前卵母细胞的发育能力较低。

从成人和前教皇捐赠者那里获得成功冷冻保存玻璃化卵母细胞的可能性将促进基础研究和商业应用。从前幼年捐赠者那里收集的卵母细胞的振动也允许缩短生成间隔,从而增加育种计划的遗传收益。然而,低温保存后发育潜力的丧失使得哺乳动物卵母细胞可能是冷冻保存最困难的细胞类型之一。在现有的冷冻保存技术中,玻璃化被广泛应用于动物和人类卵母细胞。尽管这项技术最近取得了进步,但暴露于高浓度的低温保护剂以及令人心寒的损伤和渗透应激仍然会导致一些结构和分子变化,并降低哺乳动物卵母细胞的发育潜力。在这里,我们描述了从青少年和成人捐赠者那里收集的羊卵母细胞的体外化协议,并在冷冻保存之前在体外成熟。该协议包括从卵母细胞体外成熟到体外成熟、变暖和后加热潜伏期的所有程序。在MII阶段振动的卵母细胞确实可以在变暖后受精,但它们需要额外的时间在受精前恢复由于冷冻保存程序造成的损害,并增加其发育潜力。因此,暖化后的文化条件和时间是恢复卵母细胞发育潜力的关键步骤,特别是当卵母细胞从青少年捐赠者那里收集时。

Introduction

长期储存雌性卵母细胞可以提供广泛的应用,如通过基因选择计划改善家畜繁殖,通过原地野生动物物种保护计划促进保护生物多样性,以及通过将储存的卵母细胞纳入体外胚胎生产或核移植计划1、2、3,促进体外生物技术的研究和应用。幼细胞振动也会通过缩短育种计划4的生成间隔来增加遗传收益。卵母细胞的超快速冷却和变暖的振动目前被认为是家畜卵母细胞冷冻保存的标准方法5。在反胃动物中,在体外化之前,卵母细胞通常是在体外成熟,从从屠宰场衍生的卵巢2中获取卵泡后。成人,特别是前腹卵巢4,6,确实可以提供几乎无限数量的卵母细胞冷冻保存。

在牛群中,在卵母细胞振动和变暖之后,在过去十年中,几个实验室通常报告的爆破细胞产量为>10%。然而,在小反胃动物卵母细胞化仍然被认为是相对较新的青少年和成人卵母细胞,和羊卵母细胞振动的标准方法仍有待确定2,5。尽管最近取得了进步,但玻璃化和加热的卵母细胞确实呈现出一些功能和结构上的改变,限制了它们的发展潜力7、8、9。因此,很少有文章报告在玻璃化/加热羊卵母细胞2的爆炸性发育在10%或以上。已研究了几种方法,以减少上述变化:优化玻璃化和解冻解决方案的组成10,11:试验使用不同的低温设备8,12,13:并在体外成熟(IVM)4、14、15和/或在变暖6后的恢复时间应用特定治疗。

在这里,我们描述了从青少年和成人捐赠者那里收集的羊卵母细胞的体外化协议,并在冷冻保存之前在体外成熟。该协议包括从卵母细胞体外成熟到体外化、变暖和后暖化培养期的所有程序。

Protocol

根据欧洲联盟第86/609/EC号指令和欧洲共同体委员会2007/526/EC的建议,动物议定书和下文所述的执行程序符合萨萨里大学生效的道德准则。 1. 为卵母细胞操纵准备介质 通过补充杜尔贝科的磷酸盐缓冲盐水0.1克/升青霉素和0.1克/升链霉素(PBS),为收集的卵巢的运输准备媒介。 通过稀释 9.5 克组织培养介质 (TCM) 199 粉末,用 1 升米利 Q 水补充青霉素 (0.1%) 来为?…

Representative Results

与成人捐赠者相比,青少年捐献者对卵母细胞的低温率较低。观察到的第一个效果是与成年卵母细胞相比,升温后的存活率较低(图1A:χ 2测试P<0.001)。幼细胞在变暖后表现出较低的膜完整性(图1B)。在成熟介质中使用树脂是为了验证这种糖是否能减少幼细胞的低温伤害。数据表明,23个卵母细胞成熟24小时,补充三甲糖素?…

Discussion

家畜卵母细胞低温保存不仅能长期保护雌性遗传资源,而且能促进胚胎生物技术的发展。因此,制定卵母细胞振动的标准方法将有利于牲畜和研究部门。在本议定书中,提出了一套完整的成年绵羊卵母细胞振动方法,可以代表为少年卵母细胞开发高效的卵母细胞振动系统的坚实起点。

建议方法的主要优点之一是它包括从卵母细胞收集、体外成熟、体外化和变暖的所有步骤。?…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

作者没有获得这项工作的具体资金。玛丽亚·格拉齐亚·卡帕伊教授和瓦莱里亚·帕修博士因视频画外音和在视频制作过程中建立实验室而获得感谢。

Materials

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

Referenzen

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

Play Video

Diesen Artikel zitieren
Succu, S., Serra, E., Gadau, S., Varcasia, A., Berlinguer, F. Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep. J. Vis. Exp. (173), e62272, doi:10.3791/62272 (2021).

View Video