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Luis Águila, Ricardo Felmer, María Elena Arias, Felipe Navarrete, David Martin-Hidalgo, Hoi Chang Lee, Pablo Visconti, and Rafael Fissore

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whether in vitro maturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI, in vivo-matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, although in vitro-matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation by in vitro-matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu of in vitro-matured oocytes and to replicate the molecular changes associated with in vivo capacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species.