Artificial oocyte activation: physiological,pathophysiological and ethical aspects

Infertile couples with low oocyte yield in combination with abnormal semen parameters may experience intra-cytoplasmic sperm injection (ICSI) failure. An established factor associated with ICSI failure is oocyte activation deficiency (AOD). The latter originates from seminal contributors, such as phospholipase C-zeta (PLCζ) that is not adequate to produce calcium (Ca²⁺) oscillations for oocyte activation. Apart from this natural activator, other stimulants, such as A23187, ionomycin, strontium chloride or even electric pulses, have been used in embryological laboratories to overcome AOD and ICSI failure. The aim of the present narrative review is to discuss the role of Ca⁺² oscillations in oocyte activation and summarize the evidence concerning the use of oocyte activators as agents for artificial oocyte activation (AOA). Studies in humans and animals have emerged many physiological, pathophysiological and ethical aspects of AOA. In conclusion, in mammalian eggs, the cytosolic Ca⁺² oscillations derive from a periodic release of Ca⁺² from intracellular pools. PLCζ, as well as artificial stimulants, have been used to produce Ca⁺² oscillations for AOA. As the latter may increase the risk of epigenetic induced malformations, further studies are required to clarify whether AOA constitutes an effective and safe method to overcome ICSI failure.

Abbreviations: AOA: artificial oocyte activation; AOD: oocyte activation deficiency; Ca⁺²: Calcium; CAMKII: Ca⁺²/calmodulin-dependent protein kinase II; CICR: calcium-induced calcium-release; DAG: diacylglycerol; GM-CSF: granulocyte-macrophage colony-stimulating factor; ICSI: intra-cytoplasmic sperm injection; InsP₃R: inositol-trisphosphate receptor; IP₃: inositol 1,4,5-trisphosphate; IVF: in vitro fertilization; MAP: mitogen-activated protein; MII: metaphase II; NADP: nicotinic acid adenine dinucleotide phosphate; NO: nitric oxide; PAWP: post-acrosomal WW-binding domain protein; PIP₂: phosphatidylinositol 4,5-bisphosphate; PLC: phospholipase C; PLCζ: phospholipase C-zeta; SOAFs: spermatozoon-released oocyte-activating factors; Sr⁺²: strontium; TFF: total fertilization failure     

Aspects cliniques pratiques de la vitrification ovocytaire en oncofertilité

Oocyte vitrification is a preservation fertility strategy, which can be performed in women after puberty to preserve gametes before beginning a gonadotoxic anticancer treatment. Based on available literature and our personal data, we aim to provide an overview about the feasibility, the clinical and logistic difficulties of oocyte vitrification in the field of oncofertility: limit age for oocyte cryopreservation, time required and protocols for ovarian controlled stimulation, ovarian response to stimulation, for what hopes of pregnancy?     

体外培养的人卵母细胞的超微结构

目的探讨体外培养的人卵母细胞的超微结构。方法用光镜观察体外培养前生发泡期（GV期）、培养后生发泡破裂但仍处于MⅠ期的未成熟卵母细胞和达到MⅡ期的成熟卵母细胞的形态，并将成熟卵母细胞分为优质与非优质；用透射电镜观察卵母细胞的超微结构。结果GV期卵母细胞微绒毛短小稀少；核偏一侧，核仁致密。培养后卵母细胞微绒毛粗大增多，细胞器丰富，高尔基体周围有许多分泌小泡；线粒体和脂滴伴行；卵丘细胞的胞质突起与卵母细胞的微绒毛问形成缝隙连接，卵丘细胞之间形成较多桥粒。成熟的卵母细胞粗面内质网、高尔基体减少，滑面内质网发达，线粒体幼稚化，脂滴融合，皮质颗粒沿质膜下呈线性排列，细胞连接减少，第一极体含多种细胞器。非优质卵母细胞线粒体大量存在膜和嵴模糊、膨大的现象，有的极体碎裂，透明带形态不正常，其周围卵丘细胞的凋亡率较高。结论揭示了体外培养的不同时期、不同质量的人卵母细胞的超微结构。     

点突变Uchl1不影响小鼠卵母细胞成熟

目的 通过观察突变蛋白与野生蛋白过量对卵母细胞成熟过程的影响,分析Uchl1在小鼠卵母细胞离体成熟中发挥的作用及其作用方式。方法 通过原核重组蛋白技术制备点突变（C90S和I93M）和野生型Uchl1蛋白,通过蛋白微量注射技术将突变蛋白与对照蛋白注射到未成熟卵母细胞,或体外培养液中添加突变蛋白与野生型蛋白,分析野生型Uchl1过量,或突变Uchl1-I93M蛋白添加,或泛素水解酶活性缺失的Uchl1-C90S突变蛋白添加,对于卵母细胞体外成熟的影响。结果 显微注射UCHL1融合蛋白的各组之间以及与注射PBS之间, GVBD率差异没有达到统计学显著性;同时培养液中添加Uchl1的GST融合蛋白,相对于无注射对照组,也没有统计学显著差异（P>0.05）。注射GST-C90S融合蛋白组少量GV期卵母细胞体外发育为MII期卵母细胞后呈现极体偏大于对照组。I93M点突变小鼠与WT小鼠比较,GV期卵母细胞体外GVBD率无显著差异。结论 在小鼠卵母细胞中,添加外源性Uchl1,或者有毒性作用的I93M突变体,或者水解酶活性结构改变的C90S突变体,均不影响卵母细胞成熟的GVBD进程。即使构建的I93M点突变小鼠卵母细胞GVBD率无异常。但是,其水解酶活性位点突变影响极体的形成。     

卵母细胞非整倍性改变的研究进展

妊娠失败和胎儿流产的主要原因之一是卵母细胞的非整倍性改变,并且发生率随着母亲年龄的增大而增加,三体和单体（非整倍体）胚胎至少占人类妊娠的10%,对于接近生殖寿命终点的女性,发生率可能超过50%。导致非整倍体的错误几乎总是发生在卵母细胞中,但尽管进行了深入研究,潜在的分子基础仍然令人难以捉摸。现回顾最近的研究,探讨女性减数分裂中导致非整倍体的可能机制。     

Retrieval of immature oocytes from unstimulated ovaries followed by in vitro maturation and vitrification: A novel strategy of fertility preservation for breast cancer patients

Background

We report a novel fertility preservation strategy that may be useful for young breast cancer patients who present with time constraints or concerns about the effect of ovarian stimulation.

Methods

The protocol involves retrieval of immature oocyte from unstimulated ovaries followed by in vitro maturation (IVM), and vitrification of oocytes or embryos.

Results

Thirty-eight patients (age 24-45 years) underwent vitrification of oocytes (n = 18) or embryos (n = 20). The mean ages were 33.1 ± 5.0 years and 34.7 ± 4.8 years, respectively. The mean days required to complete the egg collection was 13 days. The median numbers of vitrified oocytes and embryos per retrieval were 7 (range 1-22) and 4 (range 1-13), respectively.

Conclusions

The strategy of immature oocyte retrieval without ovarian stimulation followed by IVM and oocyte or embryo vitrification, which does not increase the serum estradiol level and delay cancer treatment, represents an attractive option of fertility preservation for many breast cancer patients.     

骨形态发生蛋白4/Smad信号通路在小鼠原始卵泡卵母细胞凋亡中的调控作用

目的 探讨骨形态发生蛋白（BMP）4/Smad 信号通路在小鼠原始卵泡卵母细胞凋亡中的调控作用。方法 取出生3d的雌性昆明小鼠卵巢,采用两步酶消化法,分离纯化卵母细胞,进行原代培养。将卵母细胞分为3组:正常对照组(Con组)、添加重组人BMP4组(BMP4组)、添加BMP4和BMP4抑制剂6-{4-［2-（1-呱啶基）乙氧基］苯基}-3-（4-吡啶基）吡唑并（1,5-A）嘧啶（dorsomorphin）组（BMP4+抑制剂组)。采用TUNEL法, 检测BMP4对卵母细胞凋亡的影响;采用免疫组织化学染色与实时定量PCR,检测BMP4对卵母细胞中p-Smad1/5/8、sohlh2、c-kit及foxo3a表达的影响。采用RNA干扰技术、转染sohlh2质粒和LY294002处理,探讨BMP4/Smad 信号通路在小鼠原始卵泡卵母细胞凋亡中的调控机制。结果 TUNEL 结果显示,BMP4组凋亡卵母细胞比例明显低于Con组(P<0.05)和BMP4+抑制剂组(P<0.05);加入BMP4可明显促进细胞Smad入核,上调 sohlh2和c-kit表达,抑制foxo3a入核; 转染sohlh2 siRNA 后,可明显阻断BMP4抑制卵母细胞凋亡的作用;转染sohlh2质粒后,p-foxo3a表达量显著增加,LY294002可明显抑制sohlh2促进foxo3a磷酸化的作用。 结论 激活BMP4/Smad信号通路可抑制小鼠原始卵泡卵母细胞的凋亡,其作用机制可能是通过上调sohlh2和c-kit基因表达,进而调控foxo3a入核而实现的。