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ISSN : 2671-4639(Print)
ISSN : 2671-4663(Online)
Journal of Animal Reproduciton and Biotechnology Vol.33 No.4 pp.229-235
DOI : https://doi.org/10.12750/JET.2018.33.4.229

Inhibition of Polo-like Kinase 1 Prevents the Male Pronuclear Formation Via Alpha-tubulin Recruiting in In Vivo-fertilized Murine Embryos

Jeonghyeon Moon, Sangho Roh†
Cellular Reprogramming and Embryo Biotechnology Laboratory, Dental Research Institute, BK21, Seoul National University School of Dentistry, Seoul, Republic of Korea
Correspondence: Sangho Roh, D.V.M., Ph.D. Tel: +82-2-880-2333, Fax: +82-02-882-0228 E-mail address: sangho@snu.ac.kr
28/11/2018 13/12/2018 13/12/2018

Abstract


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 in vivo-fertilized murine embryos. Then, the localization and expression of Plk1 was observed by immunofluorescence staining. The sperm which had entered into the oocyte cytoplasm did not form male pronuclei in BI2536-treated oocytes. The BI2536-treated oocytes showed significantly lower expression of Plk1 than non-treated control group. In addition, alpha-tubulin and Plk1 gathered around sperm head in non-treated oocytes, while BI2536-treated oocytes did not show this phenomenon. The present study demonstrates that the Plk1 inhibitor, BI2536, hinders fertilization by inhibiting the formation of murine male pronucleus.



초록


    National Research Foundation of Korea
    2016R1D1A1B03931864Ministry of SMEs and Startups

    INTRODUCTION

    Polo-like kinase 1 (Plk1) is a family of serine/threonineprotein kinase 13 (STPK13) (Wolf et al., 1997). Plk1 was first founded in Drosophila in 1988 (Sunkel and Glover, 1988). Plk1 plays a variety of roles in cells during cell division (Archambault and Glover, 2009). Plk1 regulates the centrosome maturation (Lane and Nigg, 1996), chromosome adhesion (Hauf et al., 2005), kinetochore-microtubule attachment (Brennan et al., 2007), cytokinesis and mitotic entry (Sumara I et al., 2004).

    Plk1 is highly expressed in cancer cells, therefore it is considered a proto-oncogene (Li et al., 2017). Hence, Plk1 inhibitors are used to inhibit the proliferation of cancer cells (Yan et al., 2018; Wang et al., 2018). The Plk1 inhibitor, BI2536, is a highly effective molecule that inhibits the growth and proliferation of cancer cells (Merrick et al., 2018;Noor et al., 2018;Jeong et al., 2018). BI2536 was first developed by the Boehringer Ingelheim Company in 2007. Steegmaier and colleagues showed that BI2536 is a potent inhibitor of Plk1 that inhibits tumor growth both of in vitro and in vivo (Steegmaier et al., 2007). BI2536 has been approved for acceptable safety in some clinical trials. (Frost et al., 2012). However, through extensive preclinical testing is less than the rate of side effects, it is no longer used in monotherapy (Mross et al., 2012).

    In a previous study, Plk1 is an essential factor in the first mitotic division in mouse zygote (Baran et al., 2013). It regulates the cleavage of one-cell (Zhao et al., 2010), spindle formation kinetics and APC/C activation in mouse zygote (Baran et al., 2016). Plk1 plays an important role not only in the mitotic division but also in the mouse oocyte maturation (Kheilová et al., 2015).

    Recently, many research groups have demonstrated the role and function of Plk1 in the embryo of the pre-implantation stage (Zhang Z et al., 2017;Wang H. et al., 2017). However, the accurate mechanism of Plk1 in mouse pre-implantation stage embryos still remains unclear. This study investigated that the downregulation of Plk1 using BI2536 triggers the failure of fertilization through the formation of male pronucleus.

    MATERIALS AND METHODS

    1. Chemicals

    All organic and inorganic compounds were purchased from Sigma-Aldrich Korea.

    2. Collection the in vivo-fertilized embryos and animals

    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 in vivo-fertilized embryos were collected 15 h after hCG injection, and the oviducts were removed and transferred to 2 ml TCM-199 with Hepes-CZB medium (HCZB). After tearing the ampullae of the oviducts, the in vivo-fertilized embryos were released without cumulus cells. The in vivofertilized embryos were washed five times in HCZB. All animal experiments were approved under the agreement guidelines of the Institutional Animal Care and Use Committee of Seoul National University (approval number: SNU-130123-5-7 and SNU-130123-4-7)

    3. Cell culture

    Washed in vivo-fertilized embryos were cultured in KSOM medium for 3.5 to 4 days. Mouse embryonic stem cells (mESCs) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Welgene, Korea) with 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific Korea, Korea), 1% penicillin/streptomycin solution (Life Technologies), 1% non-essential amino acids (Gibco, USA), 0.1 mM and 1000 U/ml leukemia inhibitory factor (LIF; Chemicon, USA). mESCs were cultured in 0.1% gelatin coated-dishes. Mouse skin fibroblasts (mSFs) were obtained from female B6D2F1 mice. To obtain murine skin fibroblast cells, skin tissue of B6D2F1 was washed twice with phosphate-buffered saline (PBS) including 3% penicillin/streptomycin solution and cultured in DMEM with 10% FBS and 1% penicillin/streptomycin solution. All of the cells were cultured in 37°C 5% CO2 humidified incubator.

    4. Treatment of a Plk1 inhibitor, BI2536

    The in vivo-fertilized embryos were treated with 4 nM of BI2536 (Axon medchem BV, Netherlands) to inhibit Plk1 which is essential to the mitotic division. The oocytes used as the experimental groups were placed in KSOM medium with 4 nM of BI2536 and 1% DMSO for 30 min after recovery from the oviduct in a 37°C humidified incubator. The oocytes used as the control group were cultured in KSOM medium with 1% of DMSO.

    5. Immunofluorescence

    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 (α-tubulin) was detected by mouse monoclonal IgM antibodies (1:100; Santa Cruz Biotechnology, USA) and goat-anti mouse monoclonal IgM secondary antibodies (1:500; Novus, USA). DAPI staining was used to assess nuclear morphology. All of stained cells were observed by confocal microscope. The oocytes were fixed and monitored at 0 h or 5 h after treatment of BI2536 respectively. The confocal microscopy images were acquired by LSM 700 (Zeiss, Germany). The confocal microscopy images were analyzed by Zen2 BLUE edition software (Zeiss, Germany).

    6. Statistical analysis

    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). p < 0.05 was considered significant.

    RESULTS

    To confirm the expression and location of Plk1 and α-tubulin in differentiated cells, they were subjected to immunofluorescence analysis. Plk1 was not detected in mSFs which were not in the mitotic phase (Figure 1A). However, in metaphase mESCs, Plk1 was highly expressed in the centrosome parts (Figure 1B, arrows).

    The developmental rate of BI2536-treated zygotes (0.0 ± 0.0) was significantly lower than that of non-treated in vivo-fertilized embryos (75.3 ± 3.3; p<0.05; Table 1). Even BI2536-treated zygotes failed to develop in the first mitotic division. Therefore, the focus was placed on the localization and expression of Plk1 of the mouse in vivo-fertilized embryos in the following experiments.

    The immunofluorescence images showed that the oocytes were fixed immediately after the treatment of BI2536 (Figure 2A and B). α-tubulin and Plk1 were highly expressed with sperm which entered oocyte cytoplasm in non-treated in vivo-fertilized embryos (Figure 2A, arrow). However, the expression of Plk1 and α-tubulin were low at location of sperm in BI2536-treated embryos (Figure 2B, arrow). Female nuclei have been arrested in metaphase Ⅱ without significant changes in both treated and non-treated groups (Figure 2A and B, arrowheads). The intensity of Plk1 around sperm in non-treated in vivo-fertilized embryos (Non-sperm) is significantly higher than the intensity of cytosol in non-treated in vivo-fertilized embryos (Non-cytosol) and BI2536- treated in vivo-fertilized embryos (BI-cytosol) (Figure 2C).

    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).

    DISCUSSION

    In previous studies, Plk1 is known as the essential element in mitotic cell division (Kang et al., 2006). In addition to the much-studied cyclin-dependent protein kinases, it is now appreciated that Plk1 functions as a crucial component for various mitotic events. Plk1 is tightly adjusted both temporally and spatially throughout the cell cycle. Timely recruitment of Plk1 to specific subcellular structures is likely important for proper function of Plk1 at these locations.

    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 et al., 2008). In particular, inhibition of Plk1 induces mitotic catastrophe following of the spindle assembly checkpoint in lung cancer cells (Choi et al., 2015). Plk1 inhibition elevates the antineoplastic activity in prostate cancer (Shao et al., 2015), breast cancer (Ha et al., 2018), pancreatic cancer (Mao et al., 2016).

    When Plk1 and α-tubulin were observed by immunofluorescence, fully differentiated mSFs with few mitotic cleavages had the little expression of Plk1. However, actively dividing mESCs showed higher expression of Plk1 when compared with mSFs. In particular, Plk1 was located at the regions of centrosomes in metaphase of mESCs. This result has the same consequence with previous reports from others (Schmucker and Sumara, 2014;Colicino et al., 2018).

    Plk1 inhibitors are even progressing to clinical trials (Gutteridge et al., 2016). In particular, BI2536 is widely used because it has a high effect of inhibiting plk1 and its low price (Gohda et al., 2018;Cheng et al., 2018). However, this study showed that BI2536 could have a negative effect on the male pronuclear formation. We treated BI2536 at a concentration of 100 nM or more. Consequently, the BI2536-treated embryos were proceeded apoptosis pathway (data not shown). This data was used BI2536 at a lower concentration (4 nM vs. 100 nM) and time (30 min vs. 18 h) than the results of other embryo research groups (Baran, 2013;Baran et al., 2016). Nonetheless, the male pronuclei of the BI2536-treated oocytes did not form intact morphology in this study. In previous studies, inhibition of Plk1 causes aberrant spindle (Liao et al., 2018), chromosome segregation via assembly inhibition of α-tubulin (Zhang et al., 2017). In this study, Plk1 and α-tubulin gather around the sperm which entered oocyte cytoplasm in non-treated oocytes. However, it did not show concentration of Plk1 and α-tubulin in BI2536-treated oocytes. Taken together, this data suggested that the BI2536, a Plk1 inhibitor, has the potential to cause infertility via inhibiting fertilization.

    In conclusion, this study revealed that the Plk1 inhibitor BI2536 hinder fertilization by inhibiting the formation of male pronucleus via preventing α-tubulin from being normally recruited.

    ACKNOWLEDGMENTS

    This study was supported by a grant from the National Research Foundation of Korea (NRF-2016R1D1A1B03931864) and by the Technology Development Program (S2519744) funded by the Ministry of SMEs and Startups (Korea).

    Figure

    JET-33-229_F1.gif

    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.

    JET-33-229_F2.gif

    The immunofluorescence expression of Plk1, alpha-tubulin (α-tubulin) and DNA in (A) non-treated in vivo-fertilized embryos (B) BI2536-treated in vivo-fertilized embryos shortly after fertilization. Male pronuclei (arrows) and female pronuclei (arrowheads) were presented respectively. (C) The quantitative analysis data of Plk1 intensity. *p<0.05. Scale bar = 20

    JET-33-229_F3.gif

    The immunofluorescence expression of Plk1, α-tubulin, and DNA in (A) non-treated in vivo-fertilized embryos. The pronuclei were presented in arrows. (B) BI2536-treated in vivo-fertilized embryos 5 h after fertilization. The intact sperm which had entered oocyte cytoplasm were observed (arrowhead). (C) The quantitative analysis data of expression of Plk1. PB = polar body, Non = no treatment group, BI = BI2536 treatment group *p<0.05. Scale bar = 20 μm.

    Table

    Developmental rates of non-treated in vivo-fertilized embryos and BI2536-treated in vivo-fertilized embryos

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