Abstract
BackgroundThe p21-activated kinase 1 (PAK1) is essential for mitosis and plays an important role in the regulation of microtubule assembly during oocyte meiotic maturation in mice; however, little is known about its role in porcine oocytes.ResultTotal p21-activated kinase 1 (PAK1) and phosphorylated PAK1 at Thr423 (PAK1Thr423) were consistently expressed in porcine oocytes from the germinal vesicle (GV) to the second metaphase (MII) stages, but phosphorylation of histone H3 at Ser10 (H3Ser10) was only expressed after the GV stage. Immunofluorescence analysis revealed that PAK1Thr423 and H3Ser10 colocalized on chromosomes after the GV stage. Blocking of endogenous PAK1Thr423 by injecting a specific antibody decreased the phosphorylation level of H3Ser10; however, it had no impact on chromatin condensation, meiotic progression, cleavage rate of blastomeres or the rate of blastocyst formation.ConclusionPhosphorylation of PAK1Thr423 is a spontaneous activation process and the activated PAK1Thr423 can promote the phosphorylation of H3Ser10; however, this pathway is not required for meiotic maturation of porcine oocytes or early embryonic development.
Highlights
The p21-activated kinase 1 (PAK1) is essential for mitosis and plays an important role in the regulation of microtubule assembly during oocyte meiotic maturation in mice; little is known about its role in porcine oocytes
Expression and subcellular localization of PAK1Thr423 during porcine oocyte meiotic maturation To investigate the role of PAK1 and PAK1Thr423 during porcine oocyte meiotic maturation, we examined their expression by western blotting
We provide evidence for the first time that the activity of autophosphorylated PAK1Thr423 contributes to the phosphorylation of Histone H3 at Ser10 (H3Ser10) during porcine oocyte meiotic maturation
Summary
Mammalian oocytes are arrested at prophase I when the nucleus is referred to as a germinal vesicle (GV), in which the chromatin is not condensed. The literature suggests that the PAK1-histone H3 pathway is potentially involved in regulating mitotic events, such as chromatin condensation and subsequent chromosomal capture, movement and segregation [21] It is not fully understood whether PAK1-mediated phosphorylation of histone H3 is conserved in mammalian oocytes during meiosis. To analyze the localization of PAK1Thr423 and H3Ser on chromosomes, the slides were incubated with polyclonal rabbit anti-phosphorylated PAK1 (T423) antibody (1:500) and monoclonal mouse anti-phosphorylated Histone H3 (Ser10) antibody (1:250) in PBS for 1 h at 37°C. This was followed by incubation with Alexa Fluor 594-labeled goat anti-rabbit IgG (1:500 dilution) and Alexa Fluor-labeled 488 goat anti-mouse IgG secondary antibodies (Molecular Probes) for 1 h at room temperature. Data were analyzed by one-way analysis of variance (ANOVA) using SAS 9.2 software (SAS Institute, Cary, NC, USA) and P < 0.05 was considered statistically significant
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