Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan.
From the Livestock Research Institute, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Bungoono, Oita, Japan.
Hum Reprod. 2018 Jun 1;33(6):1117-1129. doi: 10.1093/humrep/dey081.
Why are many sperm required for successful fertilization of oocytes in vitro, even though fertilization occurs in vivo when only a few sperm reach the oocyte?
Creatine produced in the ovary promotes efficient fertilization in vivo; however, in vitro, creatine is not contained in the in vitro fertilization (IVF) medium.
The IVF medium enables capacitation of sperm. However, the IVF medium does not fully mimic the in vivo environment during fertilization. Consequently, fertilization in vitro is more inefficient than in the oviduct.
STUDY DESIGN, SIZE, DURATION: Follicular 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, METHODS: Glucose and creatine levels were measured using commercial enzymatic assay kits. The effect of creatine in vivo was 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.
Oviductal 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 CAUTION: In the present study, a pharmacological approach, creatine antagonist (GPA) treatment, but not a knockout mouse model, was used to understand the role of creatine in vivo. The role of creatine in fertilization processes can only be shown in a mouse model.
A 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.
为什么许多精子需要成功受精卵子在体外，即使受精发生在体内时，只有少数精子到达卵子？
在体内，卵巢产生的肌酸促进有效的受精;然而，在体外，肌酸不包含在体外受精（IVF）培养基中。
IVF 培养基使精子获能。然而，IVF 培养基并没有完全模拟受精过程中的体内环境。因此，体外受精的效率低于输卵管。
研究设计、大小、持续时间：采集卵泡和输卵管液，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）注射到激素启动的小鼠中。使用常规的 IVF 方案，将精子在含有肌酸的 IVF 培养基中预孵育，然后与 10 个排卵的卵丘-卵母细胞复合物（每个卵母细胞 1-1000 个）在 50μl 培养基液滴中共同培养。
参与者/材料、设置、方法：使用商业酶联免疫吸附测定试剂盒测量葡萄糖和肌酸水平。通过在排卵前用或不用 GPA 处理的小鼠的交配实验评估肌酸的体内作用。为了评估含有肌酸的 IVF 培养基中孵育的精子的功能，我们分析了（1）使用计算机辅助精子分析的精子运动，（2）通过 Western blot 分析的精子获能水平，以及（3）通过花生凝集素-FITC 染色的精子顶体状况。
排卵后输卵管肌酸水平显著升高。在排卵前给小鼠注射 GPA 可显著减少受精卵的数量。向 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，每卵只需 5 个精子即可实现受精，受精卵的数量明显高于不含肌酸的对照组（P<0.01）。
局限性、谨慎的原因：在本研究中，使用药理学方法（肌酸拮抗剂[GPA]处理），而不是敲除小鼠模型，来了解肌酸在体内的作用。肌酸在受精过程中的作用只能在小鼠模型中显示。
开发了一种改良的含有肌酸的 IVF 技术，表明用少量精子可显著提高受精率。这种方法有可能对人类辅助生殖技术有很大的益处，特别是对那些精子数量有限且质量良好的患者。
研究基金/利益冲突：这项工作得到了日本学术振兴会（JSPS KAKENHI）资助项目 JP24688028、JP16H05017（M.S.）和 JP15J05331（T.U.）、日本医疗研究与发展机构（AMED）（16gk0110015h0001 至 M.S.）和美国国立卫生研究院（NIH-HD-076980 至 J.S.R.）的部分支持。作者没有什么可披露的。
