Creatine enhances the duration of sperm capacitation: a novel factor for improving in vitro fertilization with small numbers of sperm.
Creatine enhances the duration of sperm capacitation: a novel factor for improving in vitro fertilization with small numbers of sperm.
复制标题
肌酸可延长精子获能的持续时间：这是改善少量精子体外受精的新因素。
DOI：
10.1093/humrep/dey081
复制
发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Shimada,Masayuki
中科院分区：
文献类型：
--
作者：
Umehara,Takashi;Kawai,Tomoko;Goto,Masaaki;Richards,JoAnneS;Shimada,Masayuki
研究问题：为什么卵母细胞在体外成功受精需要很多精子，即使只有少数精子到达卵母细胞的体内受精也是如此？概述回答：在体内，卵巢中产生的肌酸促进有效的受精;然而，在体外，体外受精（IVF）培养基中不含肌酸。然而，IVF培养液不能完全模拟受精过程中的体内环境。因此，体外受精比输卵管受精效率更低。研究设计、规模、持续时间收集卵泡液和输卵管液，然后分析肌酸和葡萄糖水平。为了确定肌酸的生理功能，将肌酸拮抗剂3-胍基丙酸（GPA）注射到经尿道致敏的小鼠中。采用常规IVF方案，精子在含肌酸的IVF培养液中预孵育，然后与10个排卵的卵丘-卵母细胞复合体（每个卵母细胞1-1000个）在50 μl培养液滴中共培养。通过交配实验评估了肌酸在体内的作用，这些实验使用了在排卵前用或不用GPA处理的小鼠。为了评估精子在含有肌酸的IVF培养液中孵育的功能，我们分析了（1）使用计算机辅助精子测定的精子活力，（2）通过蛋白质印迹分析的精子获能水平，和（3）通过花生凝集素-FITC染色的精子顶体的条件。在排卵前给小鼠注射GPA可以显著减少受精卵的数量。在IVF培养基中添加肌酸通过增加ATP水平来增强精子获能。在肌酸组中，仅用5个精子/卵母细胞即可成功受精，受精卵母细胞的数量显著高于不加肌酸的对照组（P< 0.01）。肌酸在受精过程中的作用只能在小鼠模型中显示出来。发现的更广泛的意义一种使用含肌酸培养基的改良IVF技术被开发出来，并显示出显着改善精子数量少的受精。这种方法有可能对人类辅助生殖技术非常有益，特别是对具有有限数量的优质精子的患者。研究基金/竞争兴趣（S）这项工作部分得到了日本卫生研究所（JSPS）的资助，资助号为JP 24688028，JP 16 H 05017（给M.S.），和JP 15 J 05331（T.U.），日本医学研究开发机构（AMED）（16 gk 0110015 h 0001 to M.S.），和美国国立卫生研究院（NIH-HD-076980 to J.S.R）。作者没有什么可披露的。
STUDY QUESTIONWhy are many sperm required for successful fertilization of oocytesin vitro, even though fertilization occursin vivowhen only a few sperm reach the oocyte?SUMMARY ANSWERCreatine produced in the ovary promotes efficient fertilizationin vivo; however,in vitro, creatine is not contained in thein vitrofertilization (IVF) medium.WHAT IS KNOWN ALREADYThe IVF medium enables capacitation of sperm. However, the IVF medium does not fully mimic thein vivoenvironment during fertilization. Consequently, fertilizationin vitrois more inefficient than in the oviduct.STUDY DESIGN, SIZE, DURATIONFollicular and oviductal fluids were collected and then analyzed for creatine and glucose levels. To determine the physiological functions of creatine, the creatine antagonist 3-guanidinopropionic acid (GPA) was injected into hormonally primed mice. Using conventional IVF protocols, sperm were pre-incubated in IVF medium with creatine and then co-cultured with 10 ovulated cumulus-oocyte complexes (1–1000 per oocyte) in 50 μl medium droplets.PARTICIPANTS/MATERIALS, SETTING, METHODSGlucose and creatine levels were measured using commercial enzymatic assay kits. The effect of creatinein vivowas assessed by mating experiments using mice treated with or without GPA just before ovulation. To assess the functions of sperm incubated in IVF medium containing creatine, we analyzed (1) the motility of sperm using computer-assisted sperm assay, (2) the capacitation level of sperm by western blot analyses, and (3) the condition of sperm acrosomes by peanut agglutinin lectin-FITC staining.MAIN RESULTS AND THE ROLE OF CHANCEOviductal creatine levels were significantly increased following ovulation. Injecting mice with GPA just before ovulation significantly reduced the number of fertilized oocytes. The addition of creatine to IVF medium enhanced sperm capacitation by increasing ATP levels. Successful fertilization was achieved with as few as five sperm/oocyte in the creatine group, and the number of fertilized oocytes was significantly higher than in the control without creatine (P< 0.01).LIMITATIONS, REASONS FOR CAUTIONIn the present study, a pharmacological approach, creatine antagonist (GPA) treatment, but not a knockout mouse model, was used to understand the role of creatinein vivo. The role of creatine in fertilization processes can only be shown in a mouse model.WIDER IMPLICATIONS OF THE FINDINGSA modified IVF technique using creatine-containing medium was developed and shown to markedly improve fertilization with small numbers of sperm. This approach has the potential to be highly beneficial for human assisted reproductive technologies, especially for patients with a limited number of good quality sperm.STUDY FUNDING/COMPETING INTEREST(S)This work was supported in part by JSPS KAKENHI Grant numbers JP24688028, JP16H05017 (to M.S.), and JP15J05331 (to T.U.), the Japan Agency for Medical Research and Development (AMED) (16gk0110015h0001 to M.S.), and National Institutes of Health (NIH-HD-076980 to J.S.R). The authors have nothing to disclose.