Preliminary investigation of the effects of an FCF inhibitor interference with Septin in the early stage embryos in mice

Ge,Shao‑Qin; Wu,Yin‑Ling; Zhu,Jin‑Liang; Tian,Yuan; Wang,Shu‑Song; Gao,Zhang‑Quan

doi:10.3892/mmr.2019.10072

Preliminary investigation of the effects of an FCF inhibitor interference with Septin in the early stage embryos in mice

Authors:
- Shao‑Qin Ge
- Yin‑Ling Wu
- Jin‑Liang Zhu
- Yuan Tian
- Shu‑Song Wang
- Zhang‑Quan Gao
View Affiliations

Affiliations: The Institute of Traditional Chinese Medicine of Hebei University Health Science Centre, Baoding, Hebei 071002, P.R. China, The Medical Comprehensive Laboratory Center of Hebei University Health Science Centre, Baoding, Hebei 071002, P.R. China, The Centre for Reproductive Medicine of Peking University Third Hospital, Beijing 100191, P.R. China, Key Laboratory of Family Planning and Healthy Birth, National Health and Family Planning Commission Hebei Research Institute for Family Planning, Shijiazhuang, Hebei 050071, P.R. China
Published online on: March 21, 2019 https://doi.org/10.3892/mmr.2019.10072
Pages: 4401-4406

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The objective of the present study was to investigate the effects of for chlorfenuron (FCF) interference with the septin protein on early stage embryos in mice. The 1‑cell embryos were collected and divided into an FCF interference group and a control group. The FCF interference group was cultured in FCF media and the control group was cultured in dimethyl sulphoxide media at 37˚C with 5% CO2 until the desired phase was achieved. Septin2 protein expression was detected using immunofluorescence and western blot analysis. Blastocyst α‑tubulin was stained by immunofluorescence to observe the alterations in spindles and microtubules. The rate of early embryo development into blastocysts was significantly reduced following FCF treatment (P<0.05). In the control group, septin2 was observed with a confocal microscope; septin2 was expressed in embryos at all stages and mainly in the blastomeres from the 2‑cell stage onwards, with the expression concentrated in the nuclei of the blastomeres as identified by strong fluorescence. In the FCF interference group, septin2 was weakly expressed in the nuclei of blastomeres at the 2‑ and 4‑cell stages, and in the granulated blastomeres at the 4‑ and 8‑cell stages. Expression was barely observed in and following the morula. Granulation was observed starting from the 4‑ and 8‑cell stages. Compared with the control group, the FCF interference group exhibited irregular microtubules, abnormal spindle morphology and disordered chromosome arrangement in the blastocysts. The septin2 protein was expressed throughout the early stage embryo from the 2‑cell stage to the blastocyst and localized in the nuclei of blastomeres. When the septin protein experienced interference by the FCF inhibitor, septin2 protein expression was reduced, which simultaneously resulted in abnormal embryonic development, uneven cytoplasmic division, various sizes and a reduced number of blastomeres, granulation in the blastomeres, disordered blastocyst microtubule distribution, spindle shape alterations and an abnormality of chromosome arrangement.

View Figures

View References

1	Macklon NS, Geraedts JP and Fauser BC: Conception to ongoing pregnancy: The ‘black box’ of early pregnancy loss. Hum Reprod Update. 8:333–343. 2002. View Article : Google Scholar : PubMed/NCBI
2	Lim JH, Kim MH, Han YJ, Lee DE, Park SY, Han JY, Kim MY and Ryu HM: Cell-free fetal DNA and cell-free total DNA levels in spontaneous abortion with fetal chromosomal aneuploidy. PLoS One. 8:e567872013. View Article : Google Scholar : PubMed/NCBI
3	Vullaire L, Collins V, Callaghan T, McBain J, Williamson R and Wilton L: High incidence of complex chromosome abnormality in cleavage embryos from patients with repeated implantation failure. Fertil Sterii. 87:1053–1058. 2007. View Article : Google Scholar
4	Capalbo A, Bono S, Spizzichino L, Biricik A, Baldi M, Colamaria S, Ubaldi FM, Rienzi L and Fiorentino F: Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: Insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development. Hum Reprod. 28:509–518. 2013. View Article : Google Scholar : PubMed/NCBI
5	Hartwell LH: Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis. Exp Cell Res. 69:265–276. 1971. View Article : Google Scholar : PubMed/NCBI
6	Kinoshita M: Assembly of mammalian septins. J Biochem. 134:491–496. 2003. View Article : Google Scholar : PubMed/NCBI
7	Hall PA, Todd CB, Hyland PL, McDade SS, Grabsch H, Dattani M, Hillan KJ and Russell SE: The septin-binding protein anillin is overexpressed in diverse human tumors. Clin Cancer Res. 11:6780–6786. 2005. View Article : Google Scholar : PubMed/NCBI
8	Cao L, Ding X, Yu W, Yang X, Shen S and Yu L: Phylogenetic and evolutionary analysis of the septin protein family in metazoan. FEBS Lett. 581:5526–5536. 2007. View Article : Google Scholar : PubMed/NCBI
9	Mostowy S and Cossart P: Septins: The fourth component of thecytoskeleton. Nat Rev Mol Cell Biol. 13:183–194. 2012. View Article : Google Scholar : PubMed/NCBI
10	Gladfelter AS, Pringle JR and Lew DJ: The septin cortex at the yeast mother-bud neck. Curr Opin Microbiol. 4:681–685. 2001. View Article : Google Scholar : PubMed/NCBI
11	Spiliotis ET and Nelson WJ: Here come the septins: novel polymers that coordinate intracellular functions and organization. J Cell Sci. 119:4–10. 2006. View Article : Google Scholar : PubMed/NCBI
12	Zhu JL, Lin SL, Li M, Ouyang YC, Hou Y, Schatten H and Sun QY: Septin2 is modified by SUMOylation and required for chromosome congression in mouse oocytes. Cell Cycle. 9:1607–1616. 2010. View Article : Google Scholar : PubMed/NCBI
13	Zhu J, Qi ST, Wang YP, Wang ZB, Ouyang YC, Hou Y, Schatten H and Sun QY: Septin1 is required for spindle assembly and chromosome congression in mouse oocytes. Dev Dyn. 240:2281–2289. 2011. View Article : Google Scholar : PubMed/NCBI
14	Li S, Ou XH, Wei L, Wang ZB, Zhang QH, Ouyang YC, Hou Y, Schatten H and Sun QY: Septin 7 is required for orderly meiosis in mouse oocytes. Cell Cycle. 11:3211–3218. 2012. View Article : Google Scholar : PubMed/NCBI
15	Kopecný D, Briozzo P, Popelková H, Sebela M, Koncitíková R, Spíchal L, Nisler J, Madzak C, Frébort I, Laloue M and Houba-Hérin N: Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: A structural study. Biochimie. 92:1052–11062. 2010. View Article : Google Scholar : PubMed/NCBI
16	Heasley LR and McMurray MA: Small molecule perturbations of septins. Methods Cell Biol. 136:311–319. 2016. View Article : Google Scholar : PubMed/NCBI
17	Iwase M, Okada S, Oguchi T and Toh-e A: Forchlorfenuron, a phenylurea cytokinin, disturbs septin organization in Saccharomyces cerevisiae. Genes Genet Syst. 79:199–206. 2004. View Article : Google Scholar : PubMed/NCBI
18	Hu Q, Nelson WJ and Spiliotis ET: Forchlorfenuron alters mammalian septin assembly, organization, and dynamics. J Biol Chem. 283:29563–29571. 2008. View Article : Google Scholar : PubMed/NCBI
19	Zhu JL: Septin1/2 are required for meiotic spindle assembly and chromosome congression in mouse oocytes. Chin Acad Sci. 2011.(In Chinese).
20	Reddy SK, Rape M, Margansky WA and Kirschner MW: Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation. Nature. 446:921–925. 2007. View Article : Google Scholar : PubMed/NCBI
21	Spiliotis ET, Hunt SJ, Hu Q, Kinoshita M and Nelson WJ: Epithelial polarity requires septin coupling of vesicle transport to polyglutamylated microtubules. J Cell Biol. 180:295–303. 2008. View Article : Google Scholar : PubMed/NCBI
22	Spiliotis ET, Kinoshita M and Nelson WJ: A mitotic septin scaffold required for Mammalian chromosome congression and segregation. Science. 307:1781–1785. 2005. View Article : Google Scholar : PubMed/NCBI
23	Cleveland DW, Mao Y and Sullivan KF: Centromeres and kinetochores: From epigenetics to mitotic checkpoint signaling. Cell. 112:407–421. 2003. View Article : Google Scholar : PubMed/NCBI
24	Vallen EA, Caviston J and Bi E: Roles of Hof1p, Bni1p, Bnr1p, and myo1p in cytokinesis in Saccharomyces cerevisiae. Mol Biol Cell. 11:593–611. 2000. View Article : Google Scholar : PubMed/NCBI
25	Finger FP, Kopish KR and White JG: A role for septins in cellular and axonal migration in C. elegans. Dev Biol. 261:220–234. 2003. View Article : Google Scholar : PubMed/NCBI
26	Sitz JH, Baumgärtel K, Hämmerle B, Papadopoulos C, Hekerman P, Tejedor FJ, Becker W and Lutz B: The Down syndrome candidate dual-specificity tyrosine phosphorylation-regulated kinase 1A phosphorylates the neurodegeneration-related septin 4. Neuroscience. 157:596–605. 2008. View Article : Google Scholar : PubMed/NCBI
27	Tanaka TU and Desai A: Kinetochore-microtubule interactions: The means to the end. Curr Opin Cell Biol. 20:53–63. 2008. View Article : Google Scholar : PubMed/NCBI
28	Powers AF, Franck AD, Gestaut DR, Cooper J, Gracyzk B, Wei RR, Wordeman L, Davis TN and Asbury CL: The Ndc80 kinetochore complex forms load-bearing attachments to dynamic microtubule tips via biased diffusion. Cell. 136:865–875. 2009. View Article : Google Scholar : PubMed/NCBI
29	Angelis D and Spiliotis ET: Septin mutations in human cancers. Front Cell Dev Biol. 4:1222016. View Article : Google Scholar : PubMed/NCBI
30	Kissel H, Georgescu MM, Larisch S, Manova K, Hunnicutt GR and Steller H: The Sept4 septin locus is required for sperm terminal differentiation in mice. Dev Cell. 8:353–364. 2005. View Article : Google Scholar : PubMed/NCBI
31	Connolly D, Abdesselam I, Verdier-Pinard P and Montagna C: Septin roles in tumorigenesis. Biol Chem. 392:725–738. 2011. View Article : Google Scholar : PubMed/NCBI
32	Scott M, Hyland PL, McGregor G, Hillan KJ, Russell SE and Hall PA: Multimodality expression profiling shows SEPT9 to be overexpressed in a wide range of human tumours. Oncogene. 24:4688–4700. 2005. View Article : Google Scholar : PubMed/NCBI
33	Stanbery L, D'Silva NJ, Lee JS, Bradford CR, Carey TE, Prince ME, Wolf GT, Worden FP, Cordell KG and Petty EM: High SEPT9_v1 expression is associated with poor clinical outcomes in head and neck squamous cell carcinoma. Transl Oncol. 3:239–245. 2010. View Article : Google Scholar : PubMed/NCBI
34	Cortez BA, Rezende-Teixeira P, Redick S, Doxsey S and Machado-Santelli GM: Multipolar mitosis and aneuploidy after chrysotile treatment: A consequence of abscission failure and cytokinesis regression. Oncotarget. 7:8979–8992. 2016. View Article : Google Scholar : PubMed/NCBI