Laboratory of Biochemistry and Molecular Biology of Germ Cells, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Libechov, Czech Republic.
Laboratory of RNA Biochemistry, Faculty of Science, Charles University in Prague, Praha 2, Czech Republic.
Hum Reprod. 2024 Aug 1;39(8):1752-1766. doi: 10.1093/humrep/deae126.
Which actively translated maternal transcripts are differentially regulated between clinically relevant in vitro and in vivo maturation (IVM) conditions in mouse oocytes and zygotes?
Our findings uncovered significant differences in the global transcriptome as well as alterations in the translation of specific transcripts encoding components of energy production, cell cycle regulation, and protein synthesis in oocytes and RNA metabolism in zygotes.
Properly regulated translation of stored maternal transcripts is a crucial factor for successful development of oocytes and early embryos, particularly due to the transcriptionally silent phase of meiosis.
STUDY DESIGN, SIZE, DURATION: This is a basic science study utilizing an ICR mouse model, best suited for studying in vivo maturation. In the treatment group, fully grown germinal vesicle oocytes from stimulated ovaries were in vitro matured to the metaphase II (MII) stage either as denuded without gonadotropins (IVM DO), or as cumulus-oocyte complexes (IVM COC) in the presence of 0.075 IU/ml recombinant FSH (rFSH) and 0.075 IU/ml recombinant hCG (rhCG). To account for changes in developmental competence, IVM COC from non-stimulated ovaries (IVM COC-) were included. In vivo matured MII oocytes (IVO) from stimulated ovaries were used as a control after ovulation triggering with rhCG. To simulate standard IVM conditions, we supplemented media with amino acids, vitamins, and bovine serum albumin. Accordingly, in vitro pronuclear zygotes (IMZ) were generated by IVF from IVM DO, and were compared to in vivo pronuclear zygotes (IVZ). All experiments were performed in quadruplicates with samples collected for both polyribosome fractionation and total transcriptome analysis. Samples were collected over three consecutive months.
PARTICIPANTS/MATERIALS, SETTING, METHODS: All ICR mice were bred under legal permission for animal experimentation (no. MZE-24154/2021-18134) obtained from the Ministry of Agriculture of the Czech Republic. Actively translated (polyribosome occupied) maternal transcripts were detected in in vitro and in vivo matured mouse oocytes and zygotes by density gradient ultracentrifugation, followed by RNA isolation and high-throughput RNA sequencing. Bioinformatic analysis was performed and subsequent data validation was done by western blotting, radioactive isotope, and mitotracker dye labelling.
Gene expression analysis of acquired polysome-derived high-throughput RNA sequencing data revealed significant changes (RPKM ≥ 0.2; P ≤ 0.005) in translation between in vitro and in vivo matured oocytes and respectively produced pronuclear zygotes. Surprisingly, the comparison between IVM DO and IVM COC RNA-seq data of both fractionated and total transcriptome showed very few transcripts with more than a 2-fold difference. Data validation by radioactive isotope labelling revealed a decrease in global translation bof20% in IVM DO and COC samples in comparison to IVO samples. Moreover, IVM conditions compromised oocyte energy metabolism, which was demonstrated by both changes in polysome recruitment of each of 13 mt-protein-coding transcripts as well as by validation using mitotracker red staining.
The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE241633 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241633).
LIMITATIONS, REASONS FOR CAUTION: It is extremely complicated to achieve in vivo consistency in animal model systems such as porcine or bovine. To achieve a high reproducibility of in vivo stimulations, the ICR mouse model was selected. However, careful interpretation of our findings with regard to assisted reproductive techniques has to be made by taking into consideration intra-species differences between the mouse model and humans. Also, the sole effect of the cumulus cells' contribution could not be adequately addressed by comparing IVM COC and IVM DO, because the IVM DO were matured without gonadotropin supplementation.
Our findings confirmed the inferiority of standard IVM technology compared with the in vivo approach. It also pointed at compromised biological processes employed in the critical translational regulation of in vitro matured MII oocytes and pronuclear zygotes. By highlighting the importance of proper translational regulation during in vitro oocyte maturation, this study should prompt further clinical investigations in the context of translation.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Czech Grant Agency (22-27301S), Charles University Grant Agency (372621), Ministry of Education, Youth and Sports (EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE), and Institutional Research Concept RVO67985904. No competing interest is declared.
在体外和体内成熟（IVM）条件下，哪些活跃翻译的母本转录本在小鼠卵母细胞和胚胎中存在差异调控？
我们的发现揭示了在卵母细胞和胚胎中，整体转录组以及能量产生、细胞周期调控和蛋白质合成特定转录本翻译的改变，以及 RNA 代谢的改变。
适当调节储存的母本转录本的翻译对于卵母细胞和早期胚胎的成功发育至关重要，特别是由于减数分裂的转录沉默期。
研究设计、规模、持续时间：这是一项基础科学研究，利用 ICR 小鼠模型，最适合研究体内成熟。在治疗组中，来自刺激卵巢的完全生长的生发泡卵母细胞在没有促性腺激素的情况下进行体外成熟至 MII 期（IVM DO），或者作为有 0.075 IU/ml 重组 FSH（rFSH）和 0.075 IU/ml 重组 hCG（rhCG）的卵丘-卵母细胞复合物（IVM COC）进行成熟。为了考虑发育能力的变化，包括来自非刺激卵巢的 IVM COC（IVM COC-）。来自刺激卵巢的体内成熟的 MII 卵母细胞（IVO）在 rhCG 触发排卵后用作对照。为了模拟标准 IVM 条件，我们用氨基酸、维生素和牛血清白蛋白补充培养基。相应地，通过 IVF 从 IVM DO 产生体外原核胚胎（IMZ），并与体内原核胚胎（IVZ）进行比较。所有实验均以四倍体进行，收集样本进行多核糖体分离和全转录组分析。样本采集持续了三个月。
参与者/材料、设置、方法：所有 ICR 小鼠均根据捷克共和国农业部获得的动物实验合法许可（编号 MZE-24154/2021-18134）进行繁殖。通过密度梯度超速离心，在体外和体内成熟的小鼠卵母细胞和胚胎中检测到活跃翻译（多核糖体占据）的母本转录本，随后进行 RNA 分离和高通量 RNA 测序。进行了生物信息学分析，随后通过 Western blot、放射性同位素和 Mitotracker 染料标记进行了后续数据验证。
获得的多核糖体衍生高通量 RNA 测序数据的基因表达分析显示，体外和体内成熟的卵母细胞和分别产生的原核胚胎之间的翻译存在显著变化（RPKM≥0.2；P≤0.005）。令人惊讶的是，IVM DO 和 IVM COC 分馏和总转录组的 RNA-seq 数据之间的比较显示，只有少数转录本的差异超过 2 倍。放射性同位素标记验证显示，IVM DO 和 COC 样本的整体翻译减少了 20%，与 IVO 样本相比。此外，IVM 条件还损害了卵母细胞的能量代谢，这通过 13 种 mt-蛋白编码转录本的每个转录本的多核糖体募集变化以及使用 Mitotracker red 染色进行验证得到了证明。
本出版物中讨论的数据已存入 NCBI 的基因表达综合数据库，并可通过 GEO 系列注册号 GSE241633（https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241633）访问。
局限性、谨慎的原因：在猪或牛等动物模型系统中，很难达到体内一致性。为了实现体内刺激的高重现性，选择了 ICR 小鼠模型。然而，对于辅助生殖技术，必须谨慎解释我们的发现，同时考虑到小鼠模型与人类之间的种间差异。此外，由于 IVM DO 是在没有促性腺激素补充的情况下成熟的，因此无法充分解决单独的卵丘细胞贡献的影响。
我们的研究结果证实了标准 IVM 技术与体内方法相比的劣势。它还指出了体外成熟的 MII 卵母细胞和原核胚胎中关键翻译调控过程中存在的生物过程受损。通过强调体外卵母细胞成熟过程中适当翻译调控的重要性，本研究应该促使在翻译的背景下进一步进行临床研究。
研究资金/利益冲突：这项工作得到了捷克研究机构（22-27301S）、查理大学研究机构（372621）、教育、青年和体育部（捷克卓越研究计划，02.1.01/0.0/0.0/15_003/0000460 OP RDE）和机构研究概念的支持，没有竞争利益声明。
