Cold‐induced calcium elevation triggers DNA fragmentation in immature pig oocytes

Abstract

Fluo-4 loaded immature oocytes were cooled from 30 degrees C to various lower temperatures between 20 and 10 degrees C and changes in intracellular calcium (Ca(2+)) levels were measured. Pig oocytes cooled to 14 degrees C exhibited a clear biphasic Ca(2+) rise. Lower temperatures produced similar responses, while higher temperatures did not exert any effect. The Ca(2+) response appeared to rely on ryanodine dependent stores as removal of extracellular Ca(2+) and intracytoplasmic injection of heparin did not modify cold-induced Ca(2+) elevation, while procaine or ruthenium red virtually eliminated the response. Confocal analysis of subcellular Ca(2+) distribution during cooling revealed that the ion rises sharply within the nucleus. As Ca(2+) imbalance may activate nuclear endonucleases, DNA integrity of cooled pig oocytes was evaluated by TUNEL and comet assays. Most cooled oocytes showed clear signs of DNA fragmentation. Oocytes injected with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid tetrapotassium salt (BAPTA), a Ca(2+) chelator, maintained their DNA integrity thus confirming that intracellular Ca(2+) is involved in triggering DNA fragmentation. The protective effect exerted by ruthenium red and/or procaine further confirmed this hypothesis. These data show that a moderate and transient cooling is sufficient to cause an intracellular Ca(2+) rise that leads to DNA damage. The addition of inhibitors of ryanodine dependent Ca(2+) stores may represent a valuable protective treatment to reduce chilling injuries.

Procaine Induces Cytokinesis in Horse Oocytes via a pH-Dependent Mechanism1

“…Similar observations have been reported in sea urchin eggs [65,66], in which procaine failed to trigger either calcium influx [65] or release from intracellular calcium stores [66]. Also, in pig [21,22] and cattle [23,24], the oocyte-activation-associated calcium rise could be inhibited by injecting procaine into the cytoplasm because even low concentrations (maximum 200 lM) of procaine were able to block the ryanodine receptors on the calcium channels of the cytoplasmic calcium stores. These observations reinforce the conclusion that calcium oscillations early in fertilization and the subsequent cortical reaction, as seen in horse oocytes subjected to ICSI, do not take place in the presence of procaine.…”

“…Moreover, there is some evidence that procaine is able to disregulate cytoplasmic calcium rise(s) in female gametes. In pig [21,22] and cattle [23,24] oocytes, the calcium rise could be inhibited by injecting procaine into the cytoplasm. Further downstream in the oocyte activation pathway, the calcium rise triggers the cortical granule reaction.…”

Effect of Centrifugation Treatment before Vitrification on the Viability of Porcine Mature Oocytes and Zygotes Produced In Vitro

“…Therefore, it seems that, in pigs, it is rather the cooling procedure and not the treatment with cryoprotectant(s) that triggers parthenogenetic activation. Supporting this theory, cooling to 14 C has been reported to induce elevation of intracellular calcium levels by mobilizing Ca 2+ from intracellular stores through ryanodine receptors [32]. In our recent experiments, we found that vitrification triggers similar frequency of parthenogenetic activation of IVM porcine oocytes not only in Ca 2+ -containing but also Ca 2+ -free media (Somfai et al, unpublished data).…”

Slow cooling of human oocytes: ultrastructural injuries and apoptotic status

“…To our knowledge, the possibility that cryopreservation induces an apoptotic process has been studied only in animals. In fact, temperature decrease during oocyte maturation (39), or oocyte cryopreservation (40) in cows, and cooling of mature oocytes (41) in pigs, triggers apoptosis and DNA fragmentation in surviving oocytes. Although, in the present study, assessment of DNA fragmentation and activation of caspases in F/T oocytes did not show any sign of apoptosis, this does not rule out that inheritance of a reduced complement of functional mitochondria may compromise developmental processes.…”