Background
Meiotic maturation represents a special cell cycle that consists of two consecutive M phases, without intervening S phase. After germinal vesicle breakdown (GVBD), metaphase I (MI) spindles are formed, and homologous chromosomes begin to segregate between the oocyte and the first polar body (pbI). These oocytes subsequently progress to the second round of meiosis, but they become arrested again at metaphase II (MII) stage to wait for fertilization [
1]. During this MI to MII stage transition process, the key event is to complete the homologous chromosome segregation successfully. However, this event requires the time-sensitive and spatial coordination of spindle and chromosomal dynamic events, such as accurate bipolar spindle formation and correct kinetochore-microtubule (kMT) interaction [
2,
3]. Proper chromosome segregation during eukaryotic cell division requires that kinetochores attach to opposite spindle poles (bi-orientation) so that homologous chromosomes are pulled in opposite directions in anaphase [
4]. Failure to establish kMTs correctly can lead to chromosome mis-segregation [
5]. Furthermore, the majority of aneuploidies appear to be caused by mis-segregation of a bivalent in the first meiotic division [
6,
7]. During this MI to MII stage transition, any process error can lead to the failure of oocyte meiotic maturation. Although much has been studied regarding the GV to MI stage in meiotic cell division [
8‐
11], very little is known about how these events are orchestrated during the following MI to MII meiotic progression.
Polo-like kinase 1 (Plk1) has a variety of pivotal roles in mitotic cell division [
12‐
14], including mitotic entry [
15], centrosome maturation [
16], chromosome condensation [
17], kinetochore-microtubule attachment [
15,
18] and cytokinesis [
19‐
21]. Protein expression patterns are associated with specific subcellular localization and are coupled with specific functions [
22,
23]. Plk1 consists of an N-terminal catalytic domain and a regulating domain on the C-terminus, called the Polo box domain (PBD) [
24,
25]. It is located on different subcellular structures by binding PBD and phosphorylated proteins at Thr210 [
26,
27]. In human somatic cells, inhibition of Plk1 leads to multiple mitotic defects, including the formation of abnormal spindles and misaligned chromosomes [
28]. Moreover, our previous study showed that inhibition of Plk1 resulted in misaligned chromosomes and aberrant spindle formation in pig embryos during the first mitosis, which blocked the cell cycle arrest at prometaphase [
29]. Microinjection of Plk1 antibody in GV oocytes can lead to severe spindle defects and chromosome misalignment during mouse oocyte meiotic maturation [
30]. These results suggest that Plk1 may play a conserved role in proper spindle formation and chromosome alignment during the GV-to-MI stage of oocyte meiotic maturation.
Although there have been much information about the meiotic functions of Plk1 in some “experimental or model” animal’s oocytes, such as
Xenopus [
31,
32] and mice [
8,
33], yet little is known about its detailed role in the meiotic maturation of ‘domestic animal’ species oocytes, especially in pig oocytes. In this study, the protein expression and subcellular localization of Plk1 were examined initially by indirect immunofluorescence combined with western blot analyses during the MI-to-MII transition in pig oocytes. Then, a specific inhibitor GSK461364 was used to explore the possible role of Plk1 in porcine oocytes during the MI-to-MII transition. We found that Plk1 exhibited a specific dynamic intracellular localization pattern, which is associated with the distribution of α-tubulin during the transition from MI to MII stage. Plk1 inhibition by GSK461364 affected the meiotic maturation of oocytes, resulting in most oocytes being arrested in the ATI stage with severe chromosome segregation defects. These findings suggest that Plk1 may play an indispensable role in the first meiotic division through the regulation of proper chromosome segregation during meiosis I/meiosis II transition in pig oocytes.
Methods
Antibodies and chemicals
Mouse monoclonal anti-Plk1 and rabbit monoclonal anti-Plk1 (phospho T210) antibody were obtained from Abcam (Cambridge, UK), GSK641364 inhibitor from Selleck Chemicals (Houston, Texas, USA). Anti-GAPDH mouse polyclonal antibody and anti-β-actin mouse monoclonal antibody from Yi Feixue (Nanjing, China). All other chemicals and reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) except for those specifically mentioned.
Oocyte harvest and in vitro cultures
Pig ovaries were collected from the Yuan Run (Nanjing) slaughterhouse and transported to the laboratory in 0.9% NaCl solution within 1 h. The cumulus oocyte complexes (COCs) were aspirated from 3 to 5 mm diameter follicles, and homogeneous COCs were transferred into TCM199 medium (Gibco BRL, Gaithersburg, MD, USA) [
34] under paraffin oil at 38.5 °C in a 5% CO
2 atmosphere for maturation. The oocytes were collected after being cultured for 28, 36 and 44 h, the time points at which samples reached MI, TI and MII stages [
35], respectively, for immunostaining.
GSK461364 treatment with pig oocytes
GSK461364 is an ATP competitive, highly selective Plk1 inhibitor [
36,
37]. It was diluted in a stock solution of 5 mM in DMSO and stored at −20 °C. The oocytes were divided into three groups stochastically (at least 50 oocytes per group) and then placed into TCM199 medium for 28 h when they were most likely in meiotic I stage, then a final concentration of 0.6 or 1.2 μM GSK461364 was added for the latter oocyte cultures. The control group was treated with an identical concentration of DMSO. After a total of 44 h culture, the pbI extrusion of the oocytes was examined under a stereomicroscope.
Immunofluorescent and confocal microscopy
The oocyte samples were fixed for 30 min in 4% paraformaldehyde in phosphate buffered solution (PBS) at room temperature. After being permeabilized for 8 h with 1% Triton X-100 at 37 °C, the samples were blocked in 1% BSA for 1 h and incubated with a mouse monoclonal anti-Plk1 antibody (1:100) or anti-α-tubulin-FITC antibody (1:200) overnight at 4 °C. After washing in PBS containing 0.1% Tween 20 three times, the samples were then immersed in a Cy3-labeled goat anti-mouse IgG (H + L) (Beyotime) (1:100) at room temperature for 1 h. After washing three times, the samples were incubated with microfilament dye (phalloidin-TRITC) (1:200) at room temperature for 40 min. Finally, the cells were stained with Hoechst 33,342 for 10 min and mounted onto glass slides for confocal laser-scanning microscopy imaging (Zeiss LSM700 meta, Oberkochen, Germany).
Meiotic stage evaluation
For the analysis of spindle, chromosome and microfilament morphology, the oocytes samples were incubated with mouse anti-α-tubulin-FITC antibody (1:200), Hoechst 33,342 and phalloidin-TRITC (1:200), respectively. Then cytoskeletons were examined with a confocal laser-scanning microscope. The meiotic stages evaluation in pig oocytes was conducted as previously described by Kim et al. [
38] and Swain et al. [
39]. The oocytes with a symmetric, barrel-shaped spindle structure containing broad poles and alignment of chromosomes along the metaphase plate, microfilaments were accumulated in an actin-cap structure, signaling completion of metaphase I. The oocytes with a separate spindle structure as homologues are pulled toward opposite spindle poles, microfilaments present in the surface region of oocytes, signaling completion of anaphase-telophase I. Compared to MI stage, a disproportionate cytokinesis was formed and pbI was extruded, which were identified as completion of meiosis II.
Western blot analysis
A total of 100 oocyte samples at different developmental stages were collected and frozen in 12 μL mercaptoethanol with sodium dodecyl sulfate (SDS) sample buffer. Protein samples were boiled for 5 min to dissociate before being separated by 10% SDS PAGE. Then, the samples were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA) and blocked by immersing the membrane in 5% skim milk or BSA dissolved in Tris-buffered saline Tween 20 (TBST) for 2 h at room temperature. After incubation overnight with mouse monoclonal anti-Plk1 (1:500) or rabbit monoclonal anti-Plk1 (phospho T210) antibody (1:1000) at 4 °C, the membranes were washed in TBST three times, then incubated with goat anti-mouse IgG (1:3000; Bioworld Technology, Nanjing, China) for 2 h at room temperature. Thereafter, the membranes were washed three times, and chemiluminescence reagent (1:1; Millipore, Billerica, MA) was used for visualization. Finally, the protein level was quantified by the ratio of protein and loading control (Plk1/GAPDH) or (p-Plk1/β-actin).
Statistical analysis
All of the data from three repeated experiments was analyzed using one-way ANOVA followed by Duncan’s multiple comparisons test with GraphPad Prism 5 software; The Plk1 and p-Plk1 protein level were assessed using Quantity One software. The results were presented as the means ± SEM. P < 0.05 was considered statistically significant.
Discussion
Although much is known regarding Plk1’s functions in mitotic division [
29,
40,
41], the precise underlying mechanism of Plk1 regulation in the meiotic progression of mammalian oocytes has not been thoroughly characterized, especially during the MI-to-MII transition in porcine oocytes. In this study, we explored the subcellular localization and possible functions of Plk1 in porcine oocytes during the transition from MI to MII stage. The data indicated that Plk1 inhibition apparently affects porcine oocyte meiotic progression. Furthermore, perturbation of Plk1 activity had no obvious effect on spindle assembly, but led to a failure of chromosome segregation, which blocked the cell cycle from progressing to TI stage, remaining at ATI stage. These results showed that Plk1 contributed to porcine oocyte meiotic maturation by regulating proper chromosome segregation during the MI-to-MII stage.
Initially, the expression and localization of Plk1 were assessed in porcine oocytes undergoing meiosis, and the results revealed that Plk1 was expressed and exhibited a dynamic distribution pattern during MI-to-MII stage. Plk1 appeared to accumulate at the spindle pole region at MI or MII stages while it was associated with the spindle midzone region at TI stage. This finding was consistent with previous findings that Plk1 was distributed over the spindle midbody during ATI stage in mouse oocytes [
42] and was associated with spindle poles during the formation of M-phase spindle in rat oocytes [
43]. Different Plk1 subcellular localization is commonly coupled with its specific functions in different division stages. This dynamic localization pattern of Plk1 suggested that Plk1 may be associated with the spindle organization in MI or MII stages, and involved in the stabilization of kinetochore-microtubule attachments process during the ATI stage.
BI2536-treated oocytes fail to extrude pbI and arrest at MI stage with misaligned chromosomes in mouse oocytes [
8,
33]. Another study reported that Plk1 antibody microinjection blocked the emission of polar bodies and led to arrest at MI stage with an abnormal spindle [
43]. These previous findings suggested that Plk1 is required for normal oocyte meiotic maturation during the GV-to-MI stage. In the present study, the potential roles of Plk1 during the transition from MI to MII stage were addressed. The oocyte samples were treated with a highly selective Plk1 inhibitor GSK461364 after 28 h culture in vitro when oocytes should be in MI stage. The data showed that Plk1-inhibited oocytes failed to extrude pbI, and accompanied by a significant decrease in the level of Plk1 phosphorylation. This finding suggested that GSK461364 treatment had a significantly prohibitive effect on the Plk1 activity, which led to a fail of meiotic maturation. More importantly, these Plk1-inhibited oocytes arrested at ATI stage, thus, blocking the cell cycle from progressing to TI stage, which indicated that Plk1 play an essential role during the MI-to-MII stage in porcine oocyte, especially in ATI stage. Together with the previous findings [
8,
33,
43], these results suggested that Plk1 is required for normal oocyte meiotic maturation during both the GV-to-MI and MI-to-MII stage.
Furthermore, we explored the reason why Plk1 inhibition affected meiotic maturation in pig oocytes that arrested at ATI stage. Segregation of homologous chromosomes during ATI stage is a key event in meiosis. Any errors in this process may cause aneuploidy [
44]. Recent studies have begun to shed light on alterations in Plk1 activity that cause severe spindle defects and chromosome mis-arrangement during mouse oocyte meiotic maturation [
33]. In this study, we found that inhibition of Plk1 had no obvious effect on spindle assembly during ATI stage in pig oocytes, which was inconsistent with the results where alterations in Plk1 activity caused severe spindle defects during GV-to-MI stage in mouse oocytes [
33]. This different finding suggested different regulation mechanisms of spindle assembly between GV-to-MI stage and MI-to-MII stage of oocytes meiotic division. In addition, we also found that inhibition of Plk1 severely distorted homologous chromosome segregation during ATI stage in porcine oocytes. Similarly, BI2536-treated oocytes might prevent cohesion degradation, thus delaying chromosome segregation [
8]. These results indicated that Plk1 might play a conserved role in oocytes for proper chromosome segregation during the MI-to-MII stage. These defects in homologous chromosome segregation may due to the instability of kinetochore-microtubule attachments. Plk1 inhibition leads to a failure in APC/C activation because a defect in kinetochore-microtubule attachment activates the SAC in HeLa cells [
45]. Solc et al. (2015) demonstrated that the population of unattached kinetochores was significantly increased in BI2536-treated oocytes, indicating that Plk1 activity is required for stable kinetochore-microtubule attachments [
33]. These findings suggested that Plk1 may contribute to the stabilization of kinetochore-microtubule attachments. Together with our results, Plk1 might play an indispensable role in the stabilization of kinetochore-microtubule attachments and further influence chromosome segregation during the first meiotic division in pig oocytes.
In addition, it has been speculated that kinetochore MTs facilitate chromosome segregation and prevent re-activation of the spindle checkpoint at anaphase onset [
46]. Another study showed that sister chromatid separation causes the re-engagement of the mitotic checkpoint pathway at anaphase onset [
47]. These studies are consistent with our results, and they suggested that Plk1 inhibition may cause re-activation of the spindle checkpoint in ATI stage, which may led to cell cycle stagnation. Further studies are required to determine how Plk1 can regulate proper chromosome segregation during meiosis I
/meiosis II transition in pig oocytes.
Acknowledgements
We are thankful to Zhiqiang Guan for providing porcine ovaries. We also express our gratitude to Jing Lei for his help with using confocal laser-scanning microscopy.