Department of Surgery, School of Veterinary Medicine and Animal Science, University of São Paulo, Ave. Prof. Dr. Orlando Marques Paiva, 87, CEP 05508-270, São Paulo, Brazil.
Laboratory for Applied Toxinology, CeTICS, Butantan Institute, Ave. Vital Brasil, 1500, CEP 05503-900, São Paulo, Brazil.
Theriogenology. 2022 Aug;188:156-162. doi: 10.1016/j.theriogenology.2022.04.003. Epub 2022 Jun 2.
Advances in Artificial Reproductive Technologies (ARTs) in bovine embryos to produce cloned pregnancies have been developed in the last years, however high pregnancy losses rates still present. Those rates are associated to placental morphology alterations that are majorly focused on extracellular matrix (ECM) alterations and consequently placentome hyperplasia, increased trophoblast cell migration and vascular defects. Herein, we aimed to search, at protein level, pathways altered by ART that can modify the placental development harmony. For this, we used 4-month-old control (n = 3), SDS-decellularized (n = 3) and cloned (n = 3) cotyledons for proteomic analysis. Samples were grouped by condition and were washed, lysed, urea-reduced, acetone-precipitated, DTT-educed, iodoacetamide-alkylated, trypsin digested, and C-18 column purified. At the end, 3 μg protein were loaded in Orbitrap Fusion Lumos spectrometer (ThermoScientific). Generated spectra were exported to MaxQuant software (v1.6.10.43) to produce the protein list of each sample, and the LFQ intensity were statistically analyzed by Inferno software (v.1.1.6970). After this, proteins related to ECM and cellular junction ontologies were filtered and manually annotated using DAVID Bioinformatics Resources 6.8. From 2577 identified protein sequences by MaxQuant software, 165 (7.1%) were filtered by selected ontologies. We found 10 proteins (B2M, COL6A6, FERMT3, LGALS3BP, NIBAN2, PDLIM5, PON1, PRP9, RASIP1 and SPARC) upregulated in clone, when compared to control condition. The ten pathways that enriched more proteins were: focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway, protein digestion and absorption, amoebiasis, pathways in cancer, small cell lung cancer, platelet activation, regulation of actin cytoskeleton, and proteoglycans in cancer. Functionally, detected proteins, signaling pathways and ontologies are orchestrated to permit the binucleated trophoblastic cells migration and blood vessels modelling. In conclusion, the cloned condition presents the same mechanisms as control one, however overexpression of some specific ECM proteins could be responsible to exacerbate those mechanisms and can explain all morphophysiological alterations presented in cloned pregnancies associated to high pregnancies losses rates in this condition.
近年来，在牛胚胎的人工生殖技术（ART）方面取得了进展，以产生克隆妊娠，但高妊娠丢失率仍然存在。这些比率与胎盘形态改变有关，主要集中在细胞外基质（ECM）改变以及随后的胎盘增生、滋养层细胞迁移增加和血管缺陷。在此，我们旨在从蛋白质水平上寻找被 ART 改变的通路，这些通路可能会改变胎盘的发育平衡。为此，我们使用了 4 个月大的对照组（n=3）、SDS 去细胞化组（n=3）和克隆组（n=3）的胎盘中叶进行蛋白质组学分析。样品按条件分组，然后进行洗涤、裂解、尿素还原、丙酮沉淀、DTT 还原、碘乙酰胺烷基化、胰蛋白酶消化和 C-18 柱纯化。最后，将 3μg 蛋白质加载到 Orbitrap Fusion Lumos 光谱仪（ThermoScientific）中。生成的光谱被导出到 MaxQuant 软件（v1.6.10.43）中，以生成每个样品的蛋白质列表，LFQ 强度通过 Inferno 软件（v.1.1.6970）进行统计分析。之后，使用 DAVID 生物信息学资源 6.8 过滤与细胞外基质和细胞连接本体相关的蛋白质，并进行手动注释。从 MaxQuant 软件鉴定的 2577 个蛋白质序列中，通过选择的本体论筛选出 165 个（7.1%）。我们发现 10 种蛋白质（B2M、COL6A6、FERMT3、LGALS3BP、NIBAN2、PDLIM5、PON1、PRP9、RASIP1 和 SPARC）在克隆中上调，与对照组相比。富集更多蛋白质的十个通路是：焦点粘附、细胞外基质受体相互作用、PI3K-Akt 信号通路、蛋白质消化和吸收、变形虫病、癌症通路、小细胞肺癌、血小板激活、肌动蛋白细胞骨架调节和癌症中的蛋白聚糖。功能上，检测到的蛋白质、信号通路和本体论是协调一致的，允许双核滋养层细胞的迁移和血管建模。总之，克隆条件表现出与对照组相同的机制，但某些特定的 ECM 蛋白的过表达可能导致这些机制加剧，并可以解释与高妊娠丢失率相关的克隆妊娠中存在的所有形态生理学改变。
