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. 2017 Aug 29;15(1):69.
doi: 10.1186/s12958-017-0289-7.

Plk1 is essential for proper chromosome segregation during meiosis I/meiosis II transition in pig oocytes

Zixiao Zhang  1 , Changchao Chen  1 , Liying Ma  1 , Qiuchen Yu  1 , Shuai Li  1 , Benazir Abbasi  1 , Jiayi Yang  2 , Rong Rui  1 , Shiqiang Ju  3
Affiliations

Plk1 is essential for proper chromosome segregation during meiosis I/meiosis II transition in pig oocytes

Zixiao Zhang et al. Reprod Biol Endocrinol. .

Abstract

Background: Polo-like kinase 1 (Plk1), as a characteristic regulator in meiosis, organizes multiple biological events of cell division. Although Plk1 has been implicated in various functions in somatic cell mitotic processes, considerably less is known regarding its function during the transition from metaphase I (MI) to metaphase II (MII) stage in oocyte meiotic progression.

Methods: In this study, the possible role of Plk1 during the MI-to-MII stage transition in pig oocytes was addressed. Initially, the spatiotemporal expression and subcellular localization pattern of Plk1 were revealed in pig oocytes from MI to MII stage using indirect immunofluorescence and confocal microscopy imaging techniques combined with western blot analyses. Moreover, a highly selective Plk1 inhibitor, GSK461364, was used to determine the potential role of Plk1 during this MI-to-MII transition progression.

Results: Upon expression, Plk1 exhibited a specific dynamic intracellular localization, and co-localization of Plk1 with α-tubulin was revealed in the meiotic spindle of pig oocyte during the transition from MI to MII stage. GSK461364 treatment significantly blocked the first polar body (pbI) emission in a dose-dependent manner and resulted in a failure of meiotic maturation, with a larger percentage of the GSK461364-treated oocytes arresting in the anaphase-telophase I (ATI) stage. Further subcellular structure examination results showed that inhibition of Plk1 with GSK461364 had no visible effect on spindle assembly but caused a significantly higher proportion of the treated oocytes to have obvious defects in homologous chromosome segregation at ATI stage.

Conclusions: Thus, these results indicate that Plk1 plays an essential role during the meiosis I/meiosis II transition in porcine oocytes, and the regulation is associated with Plk1's effects on homologous chromosome segregation in the ATI stage.

Keywords: Chromosome segregation; Meiosis I/meiosis II transition; Oocyte; Pig; Polo-like 1.

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Conflict of interest statement

Ethics approval and consent to participate

The animals used in this study and their care were according to the guidelines of Animal Research Institute Committee which is prescribed by Nanjing Agricultural University, China. The workers who executed the slaughtering complied with the pig slaughtering regulations (State Council of the People’s Republic of China, No. 666).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests with respect to the authorship and/or publication of this article.

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Figures

Fig. 1
Fig. 1
Dynamic distribution of the cytoskeleton during the pig MI-to-MII transition. Samples were taken at MI, TI and MII stages. Microtubules organized bipolar spindles that were symmetrical and barrel in shape, and homologous chromosomes were arranged on the equatorial plate at MI stage; α-tubulin assembled the typical spindle structure and chromosomes were observed around α-tubulin that were in the two polar regions at TI stage. One small polar body was extruded and the microtubules were organized in a bipolar, barrel-shaped structure at the cortex below the polar body, and chromosomes were arranged on the equatorial plate at the cortex in MII stage. Microfilaments frequently formed an actin cap at MI and MII stages and formed a contractile ring at ATI stage. Green, microtubules (α-tubulin); red, microfilaments; blue, chromosomes. Scale bar, 20 μm
Fig. 2
Fig. 2
Expression and subcellular localization of Plk1 in porcine oocytes during the MI-to-MII transition. (a) Expression of Plk1 was examined using western blot analysis. Plk1 was expressed in porcine oocytes during the MI-to-MII transition, and a relatively higher Plk1 protein level was detected in MI compared to ATI and MII stages; * P < 0.05; ** P < 0.01. (b) Subcellular localization of Plk1 in porcine oocytes using immunofluorescent staining. Plk1 was enriched at spindle pole regions at MI and MII stages and closely overlapped with α-tubulin leading to the barrel-shaped spindle. Plk1 was associated with the spindle midzone region at TI stage. Red, Plk1; green, spindle; blue, chromosome. Scale bar, 20 μm
Fig. 3
Fig. 3
Effect of GSK461364 treatment during MI-to-MII stage on pbI extrusion of porcine oocytes. (a) Most of the control oocytes extruded pbI and finished meiotic maturation while a significantly larger proportion of the GSK461364 treated oocytes failed to extrude pbI. Scale bar, 20 μm. (b) The rate of extruding pbI was decreased in a dose-dependent manner in GSK461364-treated groups. (c) GSK461364 treatment significantly eliminated the phosphorylation of Plk1 in porcine oocytes. ** P < 0.01; *** P < 0.001
Fig. 4
Fig. 4
Effect of GSK461364 on cell cycle progression in porcine oocytes undergoing meiotic progression. (a) Representative images of porcine oocytes after culture with or without GSK461364 from 28 to 44 h in vitro. Most of the control oocytes reached MII stage while a larger proportion of Plk1-inhibited oocytes were arrested at ATI stage. Blue, chromosome. Scale bar, 80 μm. (b) The proportion of oocytes at different stages. Most oocytes were arrested at ATI stage after Plk1 inhibition. Compared to the control group, the percentage of Plk1-inhibited oocytes that reached MII stage was sharply decreased, whereas the proportion of oocytes that were arrested at ATI stage was significantly increased. * P < 0.05; *** P < 0.001
Fig. 5
Fig. 5
Effects of GSK461364 on spindle assembly and chromosome segregation during ATI stage in porcine oocytes. In the control group, most α-tubulin assembled a normal spindle (a-a2) and homologous chromosomes were successfully separated (a-a1); GSK461364 treatment had no effect on spindle assembly (a-a5), but led to severe defects of chromosome segregation (a-a7) in porcine oocytes. (b) There was no significant difference in spindle assembly between the control and treatment groups. (c) The percentage of Plk1-inhibited oocytes with mis-segregated chromosomes was increased compared to the control oocytes. Green, spindle; blue, chromosome. Scale bar, 20 μm. *** P < 0.001
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