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Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos
Published on: February 22, 2016
Pak2 reduction induces a failure of early embryonic development in mice
Juan Zeng1,2, Nengqing Liu1,2, Yinghong Yang1,2
1Department of Obstetrics and Gynecology, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
Background:
The quality of the early embryo is vital to embryonic development and implantation. As a highly conserved serine/threonine kinase, p21-activated kinase 2 (Pak2) participates in diverse biologic processes, especially in cytoskeleton remodeling and cell apoptosis. In mice, Pak2 knock out and endothelial depletion of Pak2 showed embryonic lethality. However, the role of Pak2 in preimplantation embryos remains unelucidated.
Methods:
In the present work, Pak2 was reduced using a specific small interfering RNA in early mouse embryos, validating the unique roles of Pak2 in spindle assembly and DNA repair during mice early embryonic development. We also employed immunoblotting, immunostaining, in vitro fertilization (IVF) and image quantification analyses to test the Pak2 knockdown on the embryonic development progression, spindle assembly, chromosome alignment, oxidative stress, DNA lesions and blastocyst cell apoptosis. Areas in chromatin with γH2AX were detected by immunofluorescence microscopy and serve as a biomarker of DNA damages.
Results:
We found that Pak2 knockdown significantly reduced blastocyst formation of early embryos. In addition, Pak2 reduction led to dramatically increased abnormal spindle assembly and chromosomal aberrations in the embryos. We noted the overproduction of reactive oxygen species (ROS) with Pak2 knockdown in embryos. In response to DNA double strand breaks (DSBs), the histone protein H2AX is specifically phosphorylated at serine139 to generate γH2AX, which is used to quantitative DSBs. In this research, Pak2 knockdown also resulted in the accumulation of phosphorylated γH2AX, indicative of increased embryonic DNA damage. Commensurate with this, a significantly augmented rate of blastocyst cell apoptosis was detected in Pak2-KD embryos compared to their controls.
Conclusions:
Collectively, our data suggest that Pak2 may serve as an important regulator of spindle assembly and DNA repair, and thus participate in the development of early mouse embryos.
Insights
p21-activated kinase 2 (Pak2) is crucial for early mouse embryo development, regulating spindle assembly and DNA repair. Its reduction impairs blastocyst formation and increases apoptosis, highlighting Pak2
Area of Science:
- Developmental Biology
- Molecular Biology
- Cell Biology
Background:
- Early embryo quality is critical for successful embryonic development and implantation.
- p21-activated kinase 2 (Pak2), a serine/threonine kinase, is involved in cytoskeleton remodeling and apoptosis.
- Previous studies indicated embryonic lethality in mice with Pak2 knockout or endothelial depletion, but its role in preimplantation embryos was unknown.
Purpose of the Study:
- To elucidate the specific roles of Pak2 in preimplantation mouse embryonic development.
- To investigate the impact of Pak2 reduction on spindle assembly, DNA repair, and blastocyst formation.
Main Methods:
- Pak2 was reduced in early mouse embryos using small interfering RNA (siRNA).
- Techniques included immunoblotting, immunostaining, in vitro fertilization (IVF), and image quantification.
- DNA double-strand breaks (DSBs) were assessed by detecting phosphorylated H2AX (γH2AX) via immunofluorescence microscopy.
Main Results:
- Pak2 knockdown significantly reduced blastocyst formation rates.
- Reduced Pak2 levels led to abnormal spindle assembly, chromosomal aberrations, and increased reactive oxygen species (ROS).
- Pak2 reduction resulted in elevated γH2AX accumulation, indicating increased DNA damage, and augmented blastocyst cell apoptosis.
Conclusions:
- Pak2 plays a significant role in regulating spindle assembly and DNA repair during early mouse embryonic development.
- Pak2 is essential for maintaining genomic stability and preventing apoptosis in preimplantation embryos.
- These findings establish Pak2 as a key regulator of early embryonic development.
