STUDY QUESTION Can oocyte functionality be assessed by observing changes in their intracytoplasmic lipid droplets (LDs) profiles? SUMMARY ANSWER Lipid profile changes can reliably be detected in human oocytes; lipid changes are linked with maternal age and impaired developmental competence in a mouse model. WHAT IS KNOWN ALREADY In all cellular components, lipid damage is the earliest manifestation of oxidative stress (OS), which leads to a cascade of negative consequences for organelles and DNA. Lipid damage is marked by the accumulation of LDs. We hypothesized that impaired oocyte functionality resulting from aging and associated OS could be assessed by changes in LDs profile, hereafter called lipid fingerprint (LF). STUDY DESIGN, SIZE, DURATION To investigate if it is possible to detect differences in oocyte LF, we subjected human GV-stage oocytes to spectroscopic examinations. For this, a total of 48 oocytes derived from 26 young healthy women (under 33 years of age) with no history of infertility, enrolled in an oocyte donation program, were analyzed. Furthermore, 30 GV human oocytes from 12 women were analyzed by transmission electron microscopy (TEM). To evaluate the effect of oocytes’ lipid profile changes on embryo development, a total of 52 C57BL/6 wild-type mice and 125 Gnpat+/− mice were also used. PARTICIPANTS/MATERIALS, SETTING, METHODS Human oocytes were assessed by label-free cell imaging via coherent anti-Stokes Raman spectroscopy (CARS). Further confirmation of LF changes was conducted using spontaneous Raman followed by Fourier transform infrared (FTIR) spectroscopies and TEM. Additionally, to evaluate whether LF changes are associated with developmental competence, mouse oocytes and blastocysts were evaluated using TEM and the lipid dyes BODIPY and Nile Red. Mouse embryonic exosomes were evaluated using flow cytometry, FTIR and FT-Raman spectroscopies. MAIN RESULTS AND THE ROLE OF CHANCE Here we demonstrated progressive changes in the LF of oocytes associated with the woman’s age consisting of increased LDs size, area, and number. LF variations in oocytes were detectable also within individual donors. This finding makes LF assessment a promising tool to grade oocytes of the same patient, based on their quality. We next demonstrated age-associated changes in oocytes reflected by lipid peroxidation and composition changes; the accumulation of carotenoids; and alterations of structural properties of lipid bilayers. Finally, using a mouse model, we showed that LF changes in oocytes are negatively associated with the secretion of embryonic exosomes prior to implantation. Deficient exosome secretion disrupts communication between the embryo and the uterus and thus may explain recurrent implantation failures in advanced-age patients. LIMITATIONS, REASONS FOR CAUTION Due to differences in lipid content between different species’ oocytes, the developmental impact of lipid oxidation and consequent LF changes may differ across mammalian oocytes. WIDER IMPLICATIONS OF THE FINDINGS Our findings open the possibility to develop an innovative tool for oocyte assessment and highlight likely functional connections between oocyte LDs and embryonic exosome secretion. By recognizing the role of oocyte LF in shaping the embryo’s ability to implant, our original work points to future directions of research relevant to developmental biology and reproductive medicine. STUDY FUNDING/COMPETING INTEREST(S) This research was funded by National Science Centre of Poland, Grants: 2021/41/B/NZ3/03507 and 2019/35/B/NZ4/03547 (to G.E.P.); 2022/44/C/NZ4/00076 (to M.F.H.) and 2019/35/N/NZ3/03213 (to Ł.G.). M.F.H. is a National Agency for Academic Exchange (NAWA) fellow (GA ULM/2019/1/00097/U/00001). K.F. is a Diamond Grant fellow (Ministry of Education and Science GA 0175/DIA/2019/28). The open-access publication of this article was funded by the Priority Research Area BioS under the program “Excellence Initiative – Research University” at the Jagiellonian University in Krakow. The authors declare no competing interest. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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哺乳动物卵母细胞脂质指纹图谱中女性年龄依赖性的变化


研究问题 可以通过观察卵母细胞质内脂滴 （LDs） 谱的变化来评估卵母细胞的功能吗？总结答案 可以在人类卵母细胞中可靠地检测到脂质谱变化;在小鼠模型中，脂质变化与母亲年龄和发育能力受损有关。已知的在所有细胞成分中，脂质损伤是氧化应激 （OS） 的最早表现，它会导致对细胞器和 DNA 的一连串负面影响。脂质损伤以 LD 的积累为标志。我们假设衰老和相关 OS 导致的卵母细胞功能受损可以通过 LDs 谱的变化来评估，以下简称脂质指纹图 （LF）。研究设计、规模、持续时间 为了调查是否有可能检测到卵母细胞 LF 的差异，我们对人类 GV 期卵母细胞进行了光谱检查。为此，分析了来自 26 名没有不孕史的年轻健康女性（33 岁以下）的 48 个卵母细胞，这些女性参加了卵母细胞捐赠计划。此外，通过透射电子显微镜 （TEM） 分析了来自 12 名女性的 30 例 GV 人卵母细胞。为了评估卵母细胞脂质谱变化对胚胎发育的影响，还使用了 52 只 C57BL/6 野生型小鼠和 125 只 Gnpat+/- 小鼠。参与者/材料、设置、方法 通过相干抗斯托克斯拉曼光谱 （CARS） 的无标记细胞成像评估人类卵母细胞。使用自发拉曼、傅里叶变换红外 （FTIR） 光谱和 TEM 进一步确认 LF 变化。 此外，为了评估 LF 变化是否与发育能力相关，使用 TEM 和脂质染料 BODIPY 和尼罗红评估小鼠卵母细胞和囊胚。使用流式细胞术、FTIR 和 FT-Raman 光谱评估小鼠胚胎外泌体。主要结果和机会的作用 在这里，我们展示了与女性年龄相关的卵母细胞 LF 的进行性变化，包括 LDs 大小、面积和数量的增加。在单个供体中也可以检测到卵母细胞的 LF 变异。这一发现使 LF 评估成为一种很有前途的工具，可以根据质量对同一患者的卵母细胞进行分级。接下来，我们证明了脂质过氧化和组成变化所反映的卵母细胞年龄相关变化;类胡萝卜素的积累;以及脂质双层结构特性的改变。最后，使用小鼠模型，我们表明卵母细胞中的 LF 变化与植入前胚胎外泌体的分泌呈负相关。外泌体分泌不足会破坏胚胎和子宫之间的通讯，因此可以解释高龄患者反复植入失败的原因。局限性，谨慎的原因 由于不同物种卵母细胞之间脂质含量的差异，脂质氧化和随之而来的 LF 变化的发育影响可能因哺乳动物卵母细胞而异。研究结果的更广泛意义我们的研究结果为开发一种用于卵母细胞评估的创新工具提供了可能性，并强调了卵母细胞 LDs 和胚胎外泌体分泌之间可能存在的功能联系。 通过认识到卵母细胞 LF 在塑造胚胎植入能力中的作用，我们的原始工作指出了与发育生物学和生殖医学相关的未来研究方向。研究资金/利益争夺 本研究由波兰国家科学中心资助，资助：2021/41/B/NZ3/03507 和 2019/35/B/NZ4/03547（给 G.E.P.）;2022/44/C/NZ4/00076 （发往 M.F.H.） 和 2019/35/N/NZ3/03213 （发往 Ł.G.）。MFH 是国家学术交流机构 （NAWA） 研究员 （GA ULM/2019/1/00097/U/00001）。K.F. 是钻石资助研究员 （Ministry of Education and Science GA 0175/DIA/2019/28）。本文的开放获取出版物由克拉科夫雅盖隆大学“卓越计划 - 研究型大学”计划下的优先研究领域 BioS 资助。作者声明没有竞争利益。试验注册号 N/A。