ABSTRACT
The factor associated suicide (Fas) and its ligand (FasL) signaling is an important regulatory pathway of apoptosis in mammalian follicles. However, whether apoptosis in bovine oocytes is regulated by the Fas-FasL signaling pathway remains unknown. In this study, localization of Fas and FasL in immature oocytes and FasL in cumulus cells were examined using immunofluorescence staining. In addition, exogenous FasL was added to an in vitro culture system to investigate apoptotic changes in bovine oocytes, using annexin-V and terminal uridine nick-end labeling staining, and real-time quantitative polymerase chain reaction. In this study, Fas was expressed in immature oocytes, whereas FasL was expressed in cumulus cells, but not in immature oocytes; annexin-V- and terminal uridine nick-end labeling-positive rates of oocytes treated with 2, 10, or 50 ng/mL FasL were higher than those of control oocytes (P < 0.05); and oocytes from the three treatment groups had higher expression levels of Fas and B cell lymphoma/leukemia-2 associated X than those in the control group (P < 0.05). Taken together, we concluded that the Fas-FasL signaling pathway was involved in regulation of bovine oocyte apoptosis, perhaps related to B cell lymphoma/leukemia-2 associated X upregulation.
Subject(s)
Cattle , Fas Ligand Protein/metabolism , Oocytes/physiology , bcl-2-Associated X Protein/metabolism , Animals , Annexin A5 , Apoptosis , Cumulus Cells/metabolism , Fas Ligand Protein/genetics , Female , Gene Expression Regulation , In Situ Nick-End Labeling , Protein Transport , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/physiology , Staining and Labeling , bcl-2-Associated X Protein/geneticsABSTRACT
The poor quality of oocytes may be the main reason for the low efficiency of the current in vitro embryo production. However, efforts are required to understand the mechanisms of oocyte development, which is believed to be largely regulated by apoptosis in vivo. The aim of this study was to investigate the levels of apoptosis in bovine immature oocytes with different developmental potentials and to determine whether early apoptosis in bovine oocytes is correlated with their subsequent development. Cumulus-oocyte complexes (COCs) were selected and classified into four groups according to oocyte cytoplasm and cumulus status. Early and late stages of apoptosis were detected by Annexin-V and TUNEL staining, respectively. Developmental competence was evaluated by nuclear maturation (MII) after in vitro maturation and development rates in different stages following in vitro fertilization. Meanwhile, the transcripts of Bcl-2 and Bax genes were carried out in immature oocytes by real-time RT-PCR. Results indicated that Annexin-V-positive oocytes were detected in various groups at different percentages, and Group III showed the highest positive ratio. No TUNEL-positive oocytes were found in any immature COCs. Group III oocytes demonstrated the highest nuclear maturation, cleavage, blastocyst, and hatching blastocyst rates. Meanwhile, Group III oocytes exhibited the highest Bax (initiating apoptosis) transcriptional level and the lowest Bcl-2 (preventing apoptosis) transcriptional level. Taken together, Annexin-V and quantitative PCR results indicated that early apoptosis was beneficial for developmental competence, while TUNEL staining showed that none of the immature oocytes were undergoing late-stage apoptosis. This is the first time that Bax and Bcl-2 transcripts were characterized in the immature bovine oocyte, and results indicated that the genes are good markers of early apoptosis and embryo development. This research overthrows the traditional view that oocytes undergoing apoptosis have poor developmental competence, and the findings will facilitate oocyte selection and improvement of in vitro embryo production.
