Treatment of Human Unfertilized Oocytes with Thimerosal and Ryanodine Supports the Development of Ca2+ and Pronucleus Formation: a Diagnostic Interest

Abstract

Objectives: Human oocytes exhibit repetitive rise in intracellular calcium concentration (Ca2+) in response to the optimal time for fertilization. However, subsequent sustained oscillations were not observed in some unfertilized oocytes, indicating that those oocytes failed to develop fully competent Ca2+ signalling mechanisms during in vitro culture. In this study, (i) the Ca2+ oscillations of the unfertilized oocytes response to ryanodine were tested and compared with those of unfertilized oocytes in response to thimerosal treatment, and (ii) the pronucleus formation was determined. Design: Oocytes allocated to this study were those that did not show signs of fertilization (formation of pronuclei, extrusion of the second polar body) by 44 h after in vitro insemination by IVF or ICSI, and that were still with no signs of degeneration or fragmentation. Aged oocytes have been previously shown to be usable as a model for the study of early developmental processes. Materials and Methods: All oocytes included in this study were incubated during 1 h in Medicult medium supplemented with thimerosal (0.67 mM) or with ryonodine (4 mM). Solvent controls were performed with DMSO only. For each oocyte, the studied parameters were: (i) pronucleus formation, and (ii) changes in (Ca2+) visualized with the use of the acetoxymethyl ester of fluo-3 (fluo-3-AM) in Confocal microscope analysis. The oocyte activation is defined in this study as: (i) the extrusion of the second polar body, and (ii) pronucleus formation. Results: Data according to all studied parameters are summarized in the Table. Tabled 1Oocyte (nb)Oocyte activation rate (%) oocyte (nb)Oocyte activation rate (%)IVFICSIThimerosal284%170%Ryanodine2015%3121%Control140%150% Open table in a new tab On 96 unfertilized oocytes treated either with thimerosal or ryanodine, 12 oocytes presented a pronucleus (12.5%). In spite of the absence of the pronucleus formation in unfertilized oocytes, thimerosal treatment induce a significant increase in (Ca2+). However, a slight Ca2+ response to ryanodine could be detected in unfertilized oocytes. Conclusion: The results of this study have shown that thimerosal can induce a significant increase in (Ca2+) in unfertilized oocytes in comparison with rynodine treatment. This increase is due to Ca2+ release from intracellular stores. The present results provide knowledge about the Ca2+ oscillations regarding the formation of pronucleus in unfertilized oocytes exposed to the drugs.