Strontium fails to induce Ca2+ release and activation in human oocytes despite the presence of functional TRPV3 channels.

Abstract

STUDY QUESTIONAre the transient receptor potential cation channels vanilloid 3 (TRPV3) present and able to mediate strontium (Sr2+) induced artificial activation in human oocytes?SUMMARY ANSWERSr2+ did not induce Ca2+ 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 Ca2+ rises and oocyte activation, demonstrating the channels were functional.WHAT IS KNOWN ALREADYSelective activation of TRPV3 by agonists induces Ca2+ entry and promotes mouse oocyte activation, and the absence of TRPV3 channels in mouse oocytes prevents Sr2+ mediated artificial activation. Sr2+ 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 Ca2+ flux has not been studied yet in human.STUDY DESIGN, SIZE, DURATIONThe protein distribution (n = 10) and mRNA expression level (n = 19) of the TRPV3 channels was investigated in human MII oocytes after IVM. The Sr2+ (10 mM) and TRPV3 agonists (200 μM 2-aminoethoxydiphenyl borate [2-APB] and 200 μM carvacrol)-induced Ca2+ response was analyzed in human (n = 15, n = 16 and n = 16, respectively) and mouse oocytes (n = 15, n = 19 and n = 26, respectively). The subsequent embryonic developmental potential following the parthenogenetic activation using these three agents was recorded in human (n = 10, n = 9 and n = 9, respectively) and mouse (n = 20 per agent) oocytes, by determining pronucleus, or 2-cell and blastocyst formation rates.PARTICIPANTS/MATERIALS, SETTING, METHODSMII 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 Ca2+ concentration was measured by time-lapse imaging after exposure to Sr2+ and TRPV3 agonists (2-APB and carvacrol). Artificial activation efficiency was assessed using these agents.MAIN RESULTS AND THE ROLE OF CHANCESr2+ did not promote Ca2+ 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 Ca2+ transient and activated a comparable percentage of more than half of the exposed human oocytes (P > 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 Ca2+ analysis and activation test (P < 0.001).LIMITATIONS REASONS FOR CAUTIONThe availability of fresh IVO matured oocytes in human was limited. Data from TRPV3 knockout model are not included.WIDER IMPLICATIONS OF THE FINDINGSThe benefit of clinical application using Sr2+ to overcome fertilization failure after ICSI requires further validation.STUDY FUNDING/COMPETING INTEREST(S)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.