Human oocyte expression and subcellular localization of the cell death regulator BCL2L10

Abstract

OBJECTIVE: Little is known about the regulation of apoptosis in human oocytes. The present study investigated whether BCL2L10, a regulator of cell death related to the Bcl-2 family, is expressed in human immature and mature oocytes, and which is its subcellular localization. DESIGN: mRNA samples from human oocytes at different stages of culture and from samples of cumulus cells surrounding MII oocytes were obtained. Gene expression was determined by quantitative RT-PCR, and double immunofluorescence labeling followed by confocal microscopy was carried out. MATERIALS AND METHODS: Quantitative PCR was performed with SYBR Green on a Lightcycler fluorimeter (Roche). Immature oocytes obtained from stimulated IVF cycles or mature non-cleaving oocytes (48h post-IVF or ICSI) were processed. Samples of cumulus cells dissociated from MII oocytes were also analyzed. GAPDH mRNA expression was used as a reference. For immunocytochemistry, individual oocytes were fixed with 4% paraformaldehyde and permeabilized with Triton X-100. Oocytes were then incubated in blocking buffer and treated with primary antibodies and Alexa-conjugated secondary antibodies (Molecular Probes). RESULTS: We obtained mRNA samples from pooled oocytes (n=50) at different stages of culture and from four samples of cumulus cells surrounding MII oocytes. Real-time PCR revealed abundant expression of BCL2L10 transcripts in all oocyte pools tested, but not in cumulus cells. Taking GAPDH as an internal standard, BCL2L10 mRNA levels were heterogeneous within the different samples. Using fluorescence microscopy, we confirmed constitutive expression of BCL2L10 in all oocytes (n=100) at all stages of maturation. Confocal imaging indicated that BCL2L10 fluorescence matched partly with organelles such as mitochondria and revealed gradual changes of BCL2L10 subcellular distribution during in vitro culture. In germinal vesicle (GV) stage oocytes, the pattern of the staining was diffuse within the ooplasm, with no immunolabeling of the GV. In metaphase I (MI) and MII oocytes, BCL2L10 was observed to be more concentrated in the oocyte cortex. Last, BCL2L10 partly colocalized with a conformation-altered form of the pro-apoptotic BAX protein in oocytes containing fragmented DNA. CONCLUSIONS: We report the presence of the apoptosis regulator BCL2L10 in human oocytes. Cytosolic and organelle-bound BCL2L10 was observed in morphologically intact oocytes. In dying oocytes, BCL2L10 colocalized with the activated form of endogenous BAX, a major cell death agonist.