Molecular requirements for doxorubicin-mediated death in murine oocytes

Abstract

We previously published evidence that oocytes exposed to doxorubicin (DXR), a widely used chemotherapeutic agent, rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis. In this report, we elucidated the molecular requirements for actions of this drug in oocytes. Our results indicate that within 1 h of exposure DXR causes rapid DNA damage, and commits the oocyte to cytoplasmic fragmentation by the fourth hour, followed by delayed oocyte activation and execution of cytoplasmic fragmentation. Inhibitors that interfere with oocyte activation consistently rescue cytoplasmic fragmentation, but fail to suppress DNA damage. There was evidence of depletion of Bax, Caspase-2, MA-3 and Bcl-x transcripts, suggesting that modulations by DXR caused recruitment of these maternal transcripts into the translation process. Furthermore, sphingolipids such as sphingosine-1-phosphate and ceramide modulate DXR actions by, respectively, altering its intracellular trafficking, or by sustaining the drug's contact with DNA.

Caspase-2 involvement during ionizing radiation-induced oocyte death in the mouse ovary

“…Indeed, a similar depletion of caspase-2 and other apoptotic mRNAs in response to genotoxic stress has previously been described in the ovulated oocyte known to be in a transcriptional repression state. 25 Moreover, our results demonstrate early activation of caspase-2 which precedes the activation of caspase-9 and -3, and pretreatment of ovaries with a selective caspase-2 inhibitor (z-VDVAD-fmk) prevents the cleavage of caspase-9 and -3 in primordial oocytes and ultimately rescues some oocytes. Taken together, our data demonstrate that caspase-2 constitutes an early step in triggering the mitochondrial apoptotic pathway in irradiated quiescent oocytes.…”

Genetic variance modifies apoptosis susceptibility in mature oocytes via alterations in DNA repair capacity and mitochondrial ultrastructure

“…Oocytes were evaluated as described [7][8][9]23 for characteristics of apoptosis, including morphological changes (e.g., cellular condensation, budding and fragmentation) and biochemical alterations (i.e., DNA cleavage using Comet Assay Kit; Trevigen, Gaithersburg, MD, USA). To primarily detect DDSB, the comet assay was performed under neutral conditions.…”

“…To primarily detect DDSB, the comet assay was performed under neutral conditions. 24 Analysis of DNA 'comets' to generate quantitative data on the percent of undamaged versus damaged DNA was performed using the VisComet program 23 (Impuls Computergestutzte Bildanalyse GmbH, Gilching, Germany). Positive controls (oocytes exposed to doxorubicin for 24 h, which have a known percentage of damaged DNA: 480%) and negative controls (freshly isolated B6C3F1 oocytes, which have intact DNA) were always run in parallel.…”

Involvement of BCL2 family members in the regulation of human oocyte and early embryo survival and death: gene expression and beyond

“…Since knocking out Mcl1 causes early lethality in mice (Rinkenberger et al 2000), a conditional knockout in the female germ line would be of great interest for a better understanding of MCL1-specific functions in female reproduction. The treatment of adult murine oocytes with the anticancer drug doxorubicin resulted in the down-regulation of BCL2L1L transcripts, accompanied by a concomitant switch in favor of its proapoptotic splicing variant BCL2L1S (BCL-XS; Jurisicova et al 2006). Additionally, the microinjection of a recombinant BCL2L1L protein in oocytes was able to inhibit doxorubicine-mediated apoptosis, suggesting that the ratio of BCL2L1L/ BCL2L1S may be critical for the oocyte to survive doxorubicin damage.…”