Lu Y, Reddy R, Ferrer Buitrago M, Vander Jeught M, Neupane J, De Vos W H, Van den Abbeel E, Lierman S, De Sutter P, Heindryckx B
Ghent-Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium.
Department of Gynecology and Obstetrics, West China Second University Hospital, Sichuan University, Renmin South Road 20, 610041 Chengdu, China.
Hum Reprod Open. 2018 Apr 9;2018(3):hoy005. doi: 10.1093/hropen/hoy005. eCollection 2018.
Are the transient receptor potential cation channels vanilloid 3 (TRPV3) present and able to mediate strontium (Sr) induced artificial activation in human oocytes?
Sr did not induce Ca rises or provoke activation in human oocytes, however, mRNA for the TRPV3 channel was present in metaphase II (MII) human oocytes after IVM and TRPV3 agonists induced Ca rises and oocyte activation, demonstrating the channels were functional.
Selective activation of TRPV3 by agonists induces Ca entry and promotes mouse oocyte activation, and the absence of TRPV3 channels in mouse oocytes prevents Sr mediated artificial activation. Sr is sometimes used to overcome fertilization failure after ICSI in the clinic, but its efficiency is still controversial and the mechanism(s) of how it mediates the Ca flux has not been studied yet in human.
The protein distribution ( = 10) and mRNA expression level ( = 19) of the TRPV3 channels was investigated in human MII oocytes after IVM. The Sr (10 mM) and TRPV3 agonists (200 μM 2-aminoethoxydiphenyl borate [2-APB] and 200 μM carvacrol)-induced Ca response was analyzed in human ( = 15, = 16 and = 16, respectively) and mouse oocytes ( = 15, = 19 and = 26, respectively). The subsequent embryonic developmental potential following the parthenogenetic activation using these three agents was recorded in human ( = 10, = 9 and = 9, respectively) and mouse ( = 20 per agent) oocytes, by determining pronucleus, or 2-cell and blastocyst formation rates.
PARTICIPANTS/MATERIALS SETTING METHODS: MII oocytes from B6D2F1 mice (6-10 weeks old) as well as human IVM oocytes and IVO oocytes (from patients aged 25-38 years old) with aggregates of smooth endoplasmic reticulum clusters were used. The expression of TRPV3 channels was determined by immunofluorescence staining with confocal microscopy and RT-PCR, and the temporal evolution of intracellular Ca concentration was measured by time-lapse imaging after exposure to Sr and TRPV3 agonists (2-APB and carvacrol). Artificial activation efficiency was assessed using these agents.
Sr did not promote Ca oscillations or provoke activation in human oocytes. Transcripts of TRPV3 channels were present in IVM MII human oocytes. TRPV3 protein was expressed and distributed throughout the ooplasm of human oocytes, rather than particularly concentrated in plasma membrane as observed in mouse MII oocytes. Both agonists of TRPV3 (2-APB and carvacrol), promoted a single Ca transient and activated a comparable percentage of more than half of the exposed human oocytes ( > 0.05). The agonist 2-APB was also efficient in activating mouse oocytes, however, significantly fewer mouse oocytes responded to carvacrol than 2-APB in both the Ca analysis and activation test ( < 0.001).
The availability of fresh IVO matured oocytes in human was limited. Data from TRPV3 knockout model are not included.
The benefit of clinical application using Sr to overcome fertilization failure after ICSI requires further validation.
STUDY FUNDING/COMPETING INTERESTS: This study was supported by FWO-Vlaanderen, China Scholarship Council and Special Research Fund from Ghent University (Bijzonder Onderzoeksfonds, BOF). No competing interests are declared.
瞬时受体电位阳离子通道香草酸亚家族成员3（TRPV3）是否存在于人类卵母细胞中，并能够介导锶（Sr）诱导的人工激活？
Sr未诱导人类卵母细胞中Ca升高或引发激活，然而，在体外成熟（IVM）后的中期II（MII）期人类卵母细胞中存在TRPV3通道的mRNA，并且TRPV3激动剂可诱导Ca升高和卵母细胞激活，证明这些通道具有功能。
激动剂对TRPV3的选择性激活可诱导Ca内流并促进小鼠卵母细胞激活，并且小鼠卵母细胞中不存在TRPV3通道可阻止Sr介导的人工激活。临床上有时使用Sr来克服卵胞浆内单精子注射（ICSI）后的受精失败，但其效率仍存在争议，并且其介导Ca通量的机制在人类中尚未得到研究。
研究设计、规模、持续时间：研究了IVM后人类MII期卵母细胞中TRPV3通道的蛋白质分布（n = 10）和mRNA表达水平（n = 19）。分析了Sr（10 mM）和TRPV3激动剂（200 μM 2-氨基乙氧基二苯硼酸盐[2-APB]和200 μM香芹酚）诱导的人类（分别为n = 15、n = 16和n = 16）和小鼠卵母细胞（分别为n = 15、n = 19和n = 26）中的Ca反应。通过确定原核、2细胞和囊胚形成率，记录了使用这三种试剂进行孤雌激活后人类（分别为n = 10、n = 9和n = 9）和小鼠（每种试剂n = 20）卵母细胞随后的胚胎发育潜能。
参与者、材料、设置、方法：使用来自B6D2F1小鼠（6 - 10周龄）的MII期卵母细胞以及具有光滑内质网簇聚集体的人类IVM卵母细胞和体外受精（IVO）卵母细胞（来自25 - 38岁的患者）。通过共聚焦显微镜免疫荧光染色和逆转录聚合酶链反应（RT-PCR）确定TRPV3通道的表达，并在暴露于Sr和TRPV3激动剂（2-APB和香芹酚）后通过延时成像测量细胞内Ca浓度的时间变化。使用这些试剂评估人工激活效率。
Sr未促进人类卵母细胞中的Ca振荡或引发激活。TRPV3通道的转录本存在于IVM的MII期人类卵母细胞中。TRPV3蛋白在人类卵母细胞的整个卵质中表达和分布，而不是像在小鼠MII期卵母细胞中那样特别集中在质膜中。两种TRPV3激动剂（2-APB和香芹酚）均促进单个Ca瞬变，并激活了超过一半的暴露人类卵母细胞中的相当比例（P > 0.05）。激动剂2-APB在激活小鼠卵母细胞方面也有效，然而，在Ca分析和激活试验中，对香芹酚有反应的小鼠卵母细胞明显少于对2-APB有反应的小鼠卵母细胞（P < 0.001）。
局限性、谨慎的原因：人类新鲜IVO成熟卵母细胞的可用性有限。未包括来自TRPV3基因敲除模型的数据。
使用Sr克服ICSI后受精失败的临床应用益处需要进一步验证。
研究资金/利益冲突：本研究得到了弗拉芒科学研究基金会（FWO）、中国国家留学基金管理委员会和根特大学特别研究基金（Bijzonder Onderzoeksfonds，BOF）的支持。未声明存在利益冲突。
