Journal of Embryo Transfer 2018; 33(4): 229-235
Published online December 29, 2018
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Jeonghyeon Moon, and Sangho Roh†
Cellular Reprogramming and Embryo Biotechnology Laboratory, Dental Research Institute, BK21, Seoul National University School of Dentistry, Seoul, Republic of Korea
Correspondence to: Correspondence: Sangho Roh, D.V.M., Ph.D. Tel:
Polo-like kinase 1 (Plk1) has been known to be a critical element in cell division including centrosome maturation, cytokinesis and spindle formation in somatic, cancer, and mammalian embryonic cells. In particular, Plk1 is highly expressed in cancer cells. Plk1 inhibitors, such as BI2536, have been widely used to prevent cell division as an anticancer drug. In this study, the fertilized murine oocytes were treated with BI2536 for 30 min after recovery from the oviduct to investigate the effect of down-regulation of Plk1 in the
Keywords: Polo-like kinase 1 (Plk1), Alpha-tubulin (α-tubulin), BI2536, Developmental rate, Fertilization
Polo-like kinase 1 (Plk1) is a family of serine/threonineprotein kinase 13 (STPK13) (Wolf
Plk1 is highly expressed in cancer cells, therefore it is considered a proto-oncogene (Li
In a previous study, Plk1 is an essential factor in the first mitotic division in mouse zygote (Baran
Recently, many research groups have demonstrated the role and function of Plk1 in the embryo of the pre-implantation stage (Zhang Z
All organic and inorganic compounds were purchased from Sigma-Aldrich Korea.Collection the
The 7.5 IU equine chorionic gonadotropin (eCG; Daesung Microbiology Lab, Korea) injected 6-8 weeks old C57BL6 X DBA2 F1-hybrid (B6D2F1) female mice (Orient Bio, Korea) by intraperitoneal for superovulation and 7.5 IU human chorionic gonadotropin (hCG; Daesung Microbiology Lab) after 48 h later. Immediately after hCG injection, female B6D2F1 mice were mated with 8-10 weeks old male B6D2F1 mice (Orient Bio, Korea). The
The oocytes, mSFs and mESCs were fixed with 4% paraformaldehyde in PBS for 30 min, then the oocytes were washed three times in PBS containing 0.5% PVP and 0.1% Triton X-100. The fixed cells were set in PBS containing 0.25% Triton X-100 for 4 h to make the membrane permeable and then incubated in PBS with 0.1% Triton X-100 and 1% BSA for blocking for 2 h in a 37°C incubator. Plk1 was detected by rabbit polyclonal IgG antibodies (1:100; Santa Cruz Biotechnology, USA) and goat-anti rabbit polyclonal IgG antibodies (1:500; Millipore, USA). Alpha-tubulin (
All experiments were iterated three times. All percentage data obtained in this study are presented as the mean ± standard deviation (S.D.). To determine the significance of differences among groups, comparisons were made using Student’s t-test as implemented in GraphPad Prism V5.0 (GraphPad Software, San Diego, CA, USA).
To confirm the expression and location of Plk1 and
The immunofluorescence images of (A) mouse skin fibrosis and (B) mouse embryonic stem cell. Polo-like kinase 1 (Plk1) localized at the regions of centrosomes (arrows). Scale bar = 5 μm.
The developmental rate of BI2536-treated zygotes (0.0 ± 0.0) was significantly lower than that of non-treated
The immunofluorescence images showed that the oocytes were fixed immediately after the treatment of BI2536 (Figure 2A and B).
The immunofluorescence expression of Plk1, alpha-tubulin (α-tubulin) and DNA in (A) non-treated
After 5 h later of treatment, the mouse zygotes formed the intact pronuclei (Figure 3A, arrows). Plk1 was not concentrated in specific region in non-treated oocytes. Instead, Plk1 spread widely in the cytosol of no treatment groups (Figure 3A). However, the expression of Plk1 was low in BI2536-treated oocytes (Figure 3B). The sperm did not form the pronuclei in BI2536-treated groups. Mouse sperm which entered oocyte cytosol remained intact (Figure 3B, arrowhead). The intensity of Plk1 in no treatment groups (Non) was significantly higher than BI2536-treated oocytes (BI) (Figure 3C).
The immunofluorescence expression of Plk1, α-tubulin, and DNA in (A) non-treated
In previous studies, Plk1 is known as the essential element in mitotic cell division (Kang
In somatic cells, the localization of Plk1 to the interphase and mitotic centromeres as cells proceed through the mitotic and meiotic cell cycle. Plk1 regulates the ultimately leads to chromosome segregation and aneuploidy during mitotic cleavage stage (Lee
When Plk1 and
Plk1 inhibitors are even progressing to clinical trials (Gutteridge
In conclusion, this study revealed that the Plk1 inhibitor BI2536 hinder fertilization by inhibiting the formation of male pronucleus via preventing