马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析
Summary
该手稿描述了一种用于形态和生物化学表征马卵母细胞的实验方法。具体来说，本文通过超声引导的卵子吸收（OPU）如何收集不成熟和成熟的马卵母细胞，以及如何调查染色体分离，纺锤形态，全局组蛋白乙酰化和mRNA表达。
Abstract
辅助生殖领域已被开发用于治疗妇女，伴侣动物和濒危物种的不育症。在马上，辅助生殖也允许从高绩效者生产胚胎，而不会中断他们的运动生涯，并有助于增加从高遗传价值的母马的数量。本手稿描述了使用卵子吸收（OPU）从马卵巢收集未成熟和成熟卵母细胞的程序。然后通过适应以前在小鼠中发展的方案，将这些卵母细胞用于研究非整倍体的发生率。具体而言，共焦激光显微镜扫描后，染色体和中期II（MII）卵母细胞的着丝粒被荧光标记并计数在连续的焦点图上。该分析显示，当从卵泡中收集未成熟卵母细胞并在体外成熟时，非整倍体率的发生率较高在体内。微管蛋白的免疫染色和特定赖氨酸残基的组蛋白4的乙酰化形式也揭示了减数分裂纺锤体的形态和组蛋白乙酰化的全局模式的差异。最后，通过逆转录和定量PCR（q-PCR）研究了编码组蛋白脱乙酰酶（HDAC）和乙酰转移酶（HAT）的mRNA的表达。 在体外和体内成熟的卵母细胞之间没有观察到转录物的相对表达的差异。与卵母细胞成熟期间转录活性的一般沉默一致，总转录物量的分析只能揭示mRNA的稳定性或降解。因此，这些研究结果表明，其他翻译和翻译后法规可能会受到影响。
总体而言，本研究描述了一种用于形态和生物化学表征马卵母细胞的实验方法由于样品可用性低，这种类型的研究极具挑战性。然而，它可以扩大我们对单调繁殖生物学和不孕症的知识。
Introduction
已经开发了广泛的辅助生殖技术来治疗妇女，伴侣动物和濒危物种的不育症。临床设置中最常见的手术之一是通过超声引导经阴道抽吸，卵子吸收（OPU） 1从卵巢卵泡中检索中期II（MII）阶段卵母细胞。然后将这些卵母细胞体外受精 （IVF），将所得的胚胎植入受体子宫。在外源性促性腺激素给药后，取出MII期（成熟）卵母细胞。然而，这种治疗在一些患者中与卵巢过度刺激综合征（OHSS）的发展有关2 。
利用完全成熟的未成熟卵母细胞（GV-stage）的内在能力自发恢复减数分裂，从卵泡中分离出来，可以获得成熟的卵母细胞，而不需要管理调理促性腺激素3 。该过程称为卵母细胞体外成熟（IVM），并且代表了辅助生殖技术的药物导向较少，成本更低且更耐用的方法。然而，胚胎发育与体外成熟卵母细胞的成功通常低于体内成熟卵母细胞4,5 。可能的解释是， 体外成熟的卵母细胞更多地受到染色体分离中的错误的影响，并且由此产生的非整倍体损害了正常的胚胎发育6 。
了解IVM期间染色体错分离的分子基础将最终披露这种技术的全部潜力。在这方面，实验方法用于研究体外成熟卵母细胞的形态和生化特征， 与体内成熟相比这里描述了卵母细胞7,8 。具体来说，使用成年和自然循环马作为实验模型说明未成熟卵母细胞的OPU和未成熟卵母细胞的IVM的程序。然后，使用免疫荧光和图像分析来研究染色体分离，纺锤形态和组蛋白乙酰化对这些配子的全局模式。最后，描述了用于mRNA表达分析的逆转录和定量PCR方案。
与啮齿动物模型相比，马不允许遗传操作，操作不太方便，需要昂贵的维护。然而，由于与人类卵巢生理学的相似性，这种模型对于卵母细胞成熟的研究正在获得相当大的兴趣，/ SUP>。此外，在马中制定IVM-IVF的可靠方案具有重大的经济利益，因为它将允许从遗传价值高的母马的数量增加。
对卵母细胞进行实验的一个局限性，特别是在单调节物种中，是限制样品的可用性。通过将以前在小鼠中开发的方法调整到马卵母细胞13,14以便进行最小化样品损失的染色体计数（参见与其他可用技术的比较的讨论），已经克服了这个限制。此外，已经优化了三重荧光染色方案以对相同的样品进行多重分析，并且仅在2个卵母细胞池上进行q-PCR分析。
总体而言，本研究描述了一种旨在形态和生物化学方面的实验方法使马卵母细胞变得更细，这种细胞类型由于样本可用性低而极具挑战性。然而，它可以扩大我们对生殖生物学和不育性不育的知识。
Protocol
Representative Results
Discussion
即使IVM已经在马上进行了超过二十年16 ，我们还不知道卵母细胞是否可以是胚胎非整倍体的起源，正如人类提出的一样。原因可能是卵母细胞扩散用于染色体计数导致相当大的样品损失。考虑到这一点，进行了调查染色体分离错误的方法的调查，以寻找适用于马卵母细胞的最合适的技术。在科学文献中发现了四种主要技术：1）染色体扩增5,18,19,2）荧光原位杂交（FISH）4,6,1…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者要感谢Fabrice Vincent对激光扫描共焦显微镜（LSCM）的支持，PhilippeBarrière和Thierry Blard进行每日超声卵巢扫描和hCG注射。这项工作部分得到“区域经济和区域伦巴第”项目“Ex Ovo Omnia”（AML授权号26096200）的支持。奖学金（2012年至2012年合约），FP7-PEOPLE-2011 CIG，研究执行机构（REA）“Pro-Ovum”（授予VL的第303640号）;“欧莱雅意大利”以及由欧洲社会基金，2007 – 2013年人力资源开发部门行动计划（合同编号：POSDRU / 89 / 1.5 / S / 62371至IM）共同资助的农业和兽医学博士后。 体内卵母细胞收集由Institut du Cheval et de l'Equitation提供资金。
Materials
| ultrasound probe | Aloka | UST-5820-7,5 | |
| human chorionic gonadotrophin | centravet | CHO004 | 1500unit/animal IV |
| detomidine | centravet | MED010 | 9-15µg/kg IV |
| butylscopolamine bromure | centravet | EST001 | 0,2mg/kg IV |
| stereomicroscope | NIKON | SMZ-2B | |
| butorphanol | centravet | DOL003 | 10µg/kg IV |
| benzyl-penicillin | centravet | DEP203 IM | 15000UI/animal |
| TCM199 | Sigma-Aldrich | M3769 10X1L | powder for hepes-buffered TCM199 |
| Hepes sodium salt | Sigma-Aldrich | H3784-100G | |
| bovine serum albumin | Sigma-Aldrich | A8806 | |
| heparin | Sigma-Aldrich | H3149-10KU | |
| NaHCO3-buffered TCM199 | Sigma-Aldrich | M2154-500ML | liquid for IVM medium |
| epidermal growth factor | Sigma-Aldrich | E4127 | |
| newborn calf serum | Sigma-Aldrich | N4762-500ML | |
| 4-well dishes | NUNCLON | 144444 | |
| incubator | Heraeus | BB6060 | |
| monastrol | Sigma-Aldrich | M8515 | |
| hyaluronidase | Sigma-Aldrich | H3506 | |
| pronase | Sigma-Aldrich | P5147 | |
| paraformaldehyde | Sigma-Aldrich | P6148 | |
| polyvinyl alcohol | Sigma-Aldrich | 341584 | |
| triton-X 100 | Sigma-Aldrich | T8787 | |
| normal donkey serum | Sigma-Aldrich | D9663 | |
| rabbit anti-Aurora B phospho-Thr232 | BioLegend | 636102 | |
| TRITC-conjugated donkey anti-rabbit IgG | Vector Laboratories | 711-025-152 | |
| Vecta-Shield | Vector Laboratories | H-1000 | |
| YOPRO1 | Thermofisher Scientific | Y3603 | |
| confocal laser scanning microscope | LSM 780 | Zeiss | |
| confocal laser scanning microscope | LSM 700 | Zeiss | |
| ImageJ software | rsb.info. nih.gov/ij/download.html | free resource | |
| mouse anti-alpha-tubulin | Sigma-Aldrich | T8203 | |
| rabbit anti-acH4K16 | Upstate Biotechnology | 07-329 | |
| AlexaFluor 488-coniugated donkey anti-mouse IgG | Life Technologies | A21202 | |
| 4’,6-diamidino-2-phenylindole | Sigma-Aldrich | D8417 | DAPI |
| centrifuge | Eppendorf | 5417R | |
| RNALater | Invitrogen | AM7020 | |
| Luciferase RNA | Promega | L4561 | |
| PicoPure RNA Isolation Kit | Applied Biosystems | 12204-01 | |
| random hexamers | Thermofisher Scientific | N8080127 | |
| mouse Moloney leukaemia virus reverse transcriptase | Thermofisher Scientific | 28025013 | |
| SYBR green supermix | BioRad | 1708880 | |
| specific primers | Sigma-Aldrich | specific primers were designed using Primer3Plus software (free resource) | |
| thermal-cycler | BioRad | MyiQ | |
| mouse monoclonal anti-CENPA | Abcam | ab13939 | |
| mouse monoclonal anti-Aurora B | Abcam | ab3609 | |
Referências
- Dellenbach, P. Transvaginal, sonographically controlled ovarian follicle puncture for egg retrieval. Lancet. 1 (8392), 1467 (1984).
- Humaidan, P. Ovarian hyperstimulation syndrome: review and new classification criteria for reporting in clinical trials. Hum Reprod. , (2016).
- Pincus, G., Enzmann, E. V. The Comparative Behavior of Mammalian Eggs in Vivo and in Vitro : I. The Activation of Ovarian Eggs. J Exp Med. 62 (5), 665-675 (1935).
- Emery, B. R., Wilcox, A. L., Aoki, V. W., Peterson, C. M., Carrell, D. T. In vitro oocyte maturation and subsequent delayed fertilization is associated with increased embryo aneuploidy. Fertil Steril. 84 (4), 1027-1029 (2005).
- Nichols, S. M., Gierbolini, L., Gonzalez-Martinez, J. A., Bavister, B. D. Effects of in vitro maturation and age on oocyte quality in the rhesus macaque Macaca mulatta. Fertil Steril. 93 (5), 1591-1600 (2010).
- Requena, A. The impact of in-vitro maturation of oocytes on aneuploidy rate. Reprod Biomed Online. 18 (6), 777-783 (2009).
- Franciosi, F. In vitro maturation affects chromosome segregation, spindle morphology and acetylation of lysine 16 on histone H4 in horse oocytes. Reprod Fertil Dev. , (2015).
- Franciosi, F. Changes in histone H4 acetylation during in vivo versus in vitro maturation of equine oocytes. Mol Hum Reprod. 18 (5), 243-252 (2012).
- Choi, Y. H., Gibbons, J. R., Canesin, H. S., Hinrichs, K. Effect of medium variations (zinc supplementation during oocyte maturation, perifertilization pH, and embryo culture protein source) on equine embryo development after intracytoplasmic sperm injection. Theriogenology. , (2016).
- Hendriks, W. K. Maternal age and in vitro culture affect mitochondrial number and function in equine oocytes and embryos. Reprod Fertil Dev. 27 (6), 957-968 (2015).
- Carnevale, E. M. The mare model for follicular maturation and reproductive aging in the woman. Theriogenology. 69 (1), 23-30 (2008).
- Ginther, O. J. The mare: a 1000-pound guinea pig for study of the ovulatory follicular wave in women. Theriogenology. 77 (5), 818-828 (2012).
- Chiang, T., Duncan, F. E., Schindler, K., Schultz, R. M., Lampson, M. A. Evidence that weakened centromere cohesion is a leading cause of age-related aneuploidy in oocytes. Curr Biol. 20 (17), 1522-1528 (2010).
- Duncan, F. E., Chiang, T., Schultz, R. M., Lampson, M. A. Evidence that a defective spindle assembly checkpoint is not the primary cause of maternal age-associated aneuploidy in mouse eggs. Biol Reprod. 81 (4), 768-776 (2009).
- Larionov, A., Krause, A., Miller, W. A standard curve based method for relative real time PCR data processing. BMC Bioinformatics. 6 (62), (2005).
- Choi, Y. H., Hochi, S., Braun, J., Sato, K., Oguri, N. In vitro maturation of equine oocytes collected by follicle aspiration and by the slicing of ovaries. Theriogenology. 40 (5), 959-966 (1993).
- Hassold, T., Hunt, P. To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet. 2 (4), 280-291 (2001).
- Akiyama, T., Nagata, M., Aoki, F. Inadequate histone deacetylation during oocyte meiosis causes aneuploidy and embryo death in mice. Proc Natl Acad Sci U S A. 103 (19), 7339-7344 (2006).
- Homer, H. A. Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin during meiosis I in mouse oocytes. Genes Dev. 19 (2), 202-207 (2005).
- Rambags, B. P. Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro. Mol Reprod Dev. 72 (1), 77-87 (2005).
- Nabti, I., Marangos, P., Bormann, J., Kudo, N. R., Carroll, J. Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity. J Cell Biol. 204 (6), 891-900 (2014).
- Shomper, M., Lappa, C., FitzHarris, G. Kinetochore microtubule establishment is defective in oocytes from aged mice. Cell Cycle. 13 (7), 1171-1179 (2014).
- Luzzo, K. M. High fat diet induced developmental defects in the mouse: oocyte meiotic aneuploidy and fetal growth retardation/brain defects. PLoS One. 7 (11), e49217 (2012).
- Ma, P., Schultz, R. M. Histone deacetylase 2 (HDAC2) regulates chromosome segregation and kinetochore function via H4K16 deacetylation during oocyte maturation in mouse. PLoS Genet. 9 (3), e1003377 (2013).
- Yang, F., Baumann, C., Viveiros, M. M., De La Fuente, R. Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte. Int J Dev Biol. 56 (10-12), 889-899 (2012).
- Luciano, A. M. Oocytes isolated from dairy cows with reduced ovarian reserve have a high frequency of aneuploidy and alterations in the localization of progesterone receptor membrane component 1 and aurora kinase B. Biol Reprod. 88 (3), 58 (2013).
- Luciano, A. M., Lodde, V., Franciosi, F., Ceciliani, F., Peluso, J. J. Progesterone receptor membrane component 1 expression and putative function in bovine oocyte maturation, fertilization, and early embryonic development. Reproduction. 140 (5), 663-672 (2010).
- Terzaghi, L. PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis. Cell Cycle. , 1-14 (2016).
- Susor, A., Jansova, D., Anger, M., Kubelka, M. Translation in the mammalian oocyte in space and time. Cell Tissue Res. 363 (1), 69-84 (2016).
- Chen, J. Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition. Genes Dev. 25 (7), 755-766 (2011).
- Ma, J., Flemr, M., Strnad, H., Svoboda, P., Schultz, R. M. Maternally recruited DCP1A and DCP2 contribute to messenger RNA degradation during oocyte maturation and genome activation in mouse. Biol Reprod. 88 (1), 11 (2013).
