PLD2 regulates microtubule stability and spindle migration in mouse oocytes during meiotic division

Animal experiments ethics and feeding condition

All the animal experiments were strictly conducted following the policies and instructions of the Care and Use of Animals in Research and Teaching and approved by the Animal Care and Use Committee of Capital Medical University with the approval No AEEI-2015-119. Mice C57BL/6- male and BALB/C-female, together with their F1 off-springs were raised as the 12 h light /dark cycle in a proper humidity and temperature with enough food and water.

Oocyte collection and culture

Ovaries were harvested from 21-day-old female F1 mice which were euthanatized with CO₂ at 44–48 h after intraperitoneal injection of 10 IU pregnant mare serum gonadotropin (PMSG, Beijing XinHuiZeAo Science and Technology). Cumulus cell-oocyte complexes (COCs) were released from ovary follicles by puncturing the ovaries with 25-gauge needles, and further cultured in Minimal Essential Medium (MEM) with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA) and 3 mg/ml bovine serum albumin (BSA, Sigma) in an atmosphere with 5% CO₂ and proper humidity at 37 °C. At 0, 2, 4, 8 and 17 h of culture, corresponding to meiotic stages at germinal vesicle (GV), germinal vesicle breakdown (GVBD), pro-metaphase I (pro-MI), metaphase I (MI) and metaphase II (MII), respectively, the cumulus cells were deposed and oocytes were processed for different experiments. As for the drug treatment, denuded oocytes were used and departed into control and drug- MEM and IVM to different periods and ready for other procedures.

Pharmacological inhibition of PLD2

In order to analyze the potential function of PLD2 in mouse oocytes, three PLD2 inhibitors were used in this study, including PLD1/2 dual inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI) (sc-300694; Santa Cruz Biotechnology, Dallas, TX, USA), and the specific PLD2 inhibitor, N-[2-[1-(3-Fluorophenyl)-4-oxo-1, 3,-8-triazaspiro [4.5] dec-8-yl] ethyl]-2-naphthalenecarboxamide (NFOT) (4171, TOCRIS Bioscience), and 1-butanol (537993; Sigma-Aldrich, St. Louis, MO, USA). FIPI binds at S757 of PLD2, which is within the HKD2 catalytic site of the enzyme, rapidly blocking in vivo PA production with subnanomolar potency. NFOT is mixed-kinetics inhibitor, binding to PLD2 at two different sites, one being at S757/S648 in HKD domain, and another being an allosteric site that is a natural phosphoinositide biding pocket in PH domain and usually occupied by PIP2. NFOT affects both PA production and PIP2 binding to PLD2. And 1-butanol is another PLD inhibitor, blocking PLD production of PA, and widely employed to identify PLD/PA-driven processes.

All solid drugs were reconstructed to stock solution at 50 mM in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA), and further diluted in oocyte culture medium to final working concentration before use. For control, same amount of DMSO was added in the medium. The quantity of DMSO was not more than 0.1% (v/v) in working solution. 1-butanol was resupplied every hour for the reduced effect.

Cooling treatment

Oocytes at GV stage were pre-cultured for 8 h in maturation medium supplemented with DMSO or NFOT, at which time, the majority oocytes were supposed to develop to MI stage. After thoroughly washed, these oocytes were placed on ice and incubated in fresh M2 medium (M5910; Sigma-Aldrich, St. Louis, MO, USA) for additional 20 min, 40 min and 60 min, respectively, and then processed for analysis of microtubules and PLD2 through immunofluorescence procedure.

Immunofluorescence

Oocytes were fixed in 1% paraformaldehyde (PFA) in PEM buffer (100 mM Pipes, 1 mM MgCl₂ and 1 mM EGTA, pH 6.9) with 0.5% Triton X-100 for 30 min at room temperature. After triple washing in phosphate-buffered saline (PBS) containing 0.2% Triton X-100 (PBST), oocytes were blocked in PBST with 10% normal goat serum, 1% BSA and 0.3 M glycine for 1 h at room temperature, and incubated in properly diluted primary antibodies, including rabbit anti-PLD2 (1:1000, HPA013397; Sigma-Aldrich, St. Louis, MO, USA), mouse anti-acetylated tubulin (1:3000, T7451; Sigma-Aldrich, St. Louis, MO, USA), mouse anti-PIP2 (1:500, sc-53412; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-phosphor Colifin (Ser3) (1:1000, GTX50199; GeneTex, Irvine, CA, USA) and mouse anti-RhoA (1:500, sc-418; Santa Cruz Biotechnology, Dallas, TX, USA), at 4 °C, overnight. After triple washing in PBST, each for 10 min, oocytes were treated with goat anti-mouse Alexa-488 (1:500; Molecular Probes) or goat anti-rabbit Alexa-594 (1:500; Molecular Probes) in a light-proof box for 45 min at room temperature. After washed as described, oocytes were mounted on the glass slides with Vectashield mounting medium containing DAPI (H-1200; Vector Laboratories). All images were taken by DP-97 Olympus microscope.

The actin staining was carried out following special procedure as described previously (Na & Zernicka-Goetz, 2006). Oocytes were exposed to the acidic Tyrode’s solution (T1788, Sigma) for 2 min to remove the zona pellucida. After short recovery in culture medium, these oocytes were fixed in the fix solution (4% PFA, 0.15% glutaraldehyde, 0.06% Triton X-100, 130 mM KCl, 25 mM HEPES and 3 mM MgCl₂, pH 6.9), for 30 min at room temperature, and then permeabilized in 0.2% Triton X-100 in PEM buffer prior to additional 10 min incubation in fix solution. After thoroughly washed, these cells were labeled with Alexa flour 555—phalloidin (1:3000, 8953; Cell Signaling Technology, Danvers, MA, USA). The samples were mounted and analyzed as described above.

Western blot

50 oocytes in each sample were collected in Laemmli lysis buffer (161-0737; Bio-Rad, Hercules, CA, USA) with protease inhibitor cocktail (P2714; Sigma-Aldrich, St. Louis, MO, USA) and stored at −80°C. Before use, the samples were degenerated at 100°C for 5 min and cooled down on ice. The proteins were separated by 10% SDS-PAGE and blotted to PVDF membrane (IPVH00010; Millipore, Billerica, MA, USA), the membranes were then blocked in 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) for 1 h at room temperature, and then incubated overnight at 4 °C in diluted primary antibodies. After washed three times, each for 15 min, the membranes were further labeled with horseradish peroxidase-conjugated secondary antibodies (ZSGB-BIO) for 45 min at room temperature. After triple washes as described, the membranes were treated with enhanced chemiluminescence (ECL) system (P1010; Applygen Technologies Inc., Beijing, China). The semi-quantitative gray scale analysis of bands was processed using Image J software.

Data statistics

All experiments were performed at least 3 replicates and 30–50 oocytes were included in each group. The statistics and graphs were processed and produced by GraphPad Prism 5.01 (GraphPad Software, La Jolla,CA, USA). Data were given as the mean ± SEM and P < 0.05 was considered significant.

Stable expression of PLD2 and its relationship with spindle in oocyte meiosis

The protein expression of PLD2 in mouse oocytes was firstly detected by Western blot analysis. As shown in Fig. 1, a high level of PLD2 protein expression was revealed in mouse oocytes; importantly, the protein level was consistently stable from germinal vesicle (GV) stage to metaphase II (MII) (Fig. 1A), and further statistical analysis of the band gray scale confirmed there was no significant difference in PLD2 level among different meiotic stages, as checked at GV, germinal vesicle breakdown (GVBD), metaphase I (MI) and MII stage, respectively (Fig. 1B).

Then, the subcellular distribution pattern of PLD2 was explored with an immunofluorescence approach. As shown in Fig. 1, no special aggregation was observed in oocytes at GV stage (Fig. 1C, 3), indicating PLD2 was evenly distributed in cytoplasm before the resumption of meiosis. Upon GVBD, as the chromatin was condensed into individual chromosomes (Fig. 1C, 5), PLD2 began to aggregate around the condensing chromosomes, simultaneously with the newly formed microtubules (Fig. 1C, 6–8). As cell cycle progressed to pro-metaphase I (pro-MI) and MI stage, microtubules were gradually organized into bi-polar spindle with all the chromosomes properly aligned on the equatorial plate, evidently, PLD2 sustained its co-localization with microtubules throughout the whole process of spindle organizing (Fig. 1C, 10–12, 14–16). The co-localization pattern lasted to the coming anaphase, during which PLD2 was overlapped with microtubules at polar area (Fig. 1C, 18–20). When oocytes developed to MII stage, PLD2 was again distributed across the re-formed spindle and precisely co-localized with microtubules (Fig. 1C, 21–24). These data strongly suggest possible involvement of PLD2 in meiotic spindle formation or positioning in mouse oocytes.

PLD2 inhibition suppressed spindle migration to the cortex during oocyte meiosis

We intended to explore the possible role of PLD2 in oocyte meiotic maturation by using a specific inhibitor to PLD2. After GV oocytes were cultured for 17 h with NFOT at 20 µM, which was a defined proper concentration after series of experiments, nearly all the oocytes were arrested at MI stage but not matured to MII, further statistical analysis demonstrated that the number of oocytes with first polar body extrusion was significantly lower in the NFOT group than that in the control (Fig. 2B). Importantly, all the MI oocytes were installed with an enlarged spindle which was center-positioned (Fig. 2A).

We went back to check spindle structure and positioning at 8 h, 10 h and 12 h of NFOT incubation. As showed in Fig. 2C, all the oocytes remained at MI stage in NFOT group (Fig. 2C, 1’–3’), no matter the incubation time extended up to 12 h, and the spindle was enlarged and located closely to the central area (Fig. 2C, 4’–6’). PLD2 was co-localized with microtubule on spindle and aggregated as big dots, distributed near to spindle poles (Fig. 2C, 7’: arrows) and randomly in cytoplastic area (Fig. 2C, 7’: arrowhead). In contrast, nearly in all control oocytes, the spindle exhibited normal size and was located in the sub-cortex position (Fig. 2C, 4–5), some oocytes developed to MII stage as culture time increased to 12 h (Fig. 2C, 3). To quantitatively compare spindle changes after NFOT treatment, we measured the value of long axis length of spindle (l), oocyte spherical radius (R) and the distance between spindle center and oocyte spherical center (d) in each oocyte. The ratio of “l” to “R” indicated the relative value of spindle length and was denoted as “L” (L, L = l / R). The ratio of “d” to “R” indicated the relative value of spindle migrating distance and was denoted as “D” (D, D = d/R) (Fig. 2D). Statistical analysis demonstrated that the “L” value was significantly higher in NFOT-treated oocytes than that in control at 8 h, 10 h and 12 h incubation (Fig. 2E) (P < 0.05), indicating the spindle was dramatically enlarged with PLD2 inhibition, at the same time, the “D” value was markedly decreased in NFOT group at all the time points (Fig. 2F) (P < 0.001), implying spindle migrating toward the cortex was suppressed in oocytes when PLD2 was inhibited with NFOT.

As the enlarged spindle, we proposed that suppression of PLD2 may change the organizing pattern of spindle microtubules, and this proposal was proved by the classic cold-induced microtubule depolymerization experiment. When MI oocytes were incubated on ice for different periods with NFOT, we found that microtubules were more stable in NFOT-treated oocytes. As showed in Fig. 3, microtubules were totally disassembled in control oocytes after 20 min incubation on ice (Fig. 3, 1), simultaneously, PLD2 assembly was also disappeared (Fig. 3, 5). However, in NFOT group, microtubules remained with PLD2 in polar area at 20 min of incubation (Fig. 3, 2, 6), even detectable at 40 min (Fig. 3, 3, 7), and totally depolymerized when checked at 60 min (Fig. 3, 4, 8). Microtubule dynamics was highly consistent with PLD2 signal during the whole process of cold treatment. These data indicate that PLD2 is associated with normal stability of microtubules, and PLD2 inhibition could promote microtubule stability, might contributing to the formation of large spindle.

Catalytic inhibition of PLD2 did not affect the cortex-towards spindle movement

PLD2 catalyzes the hydrolysis of phosphatidylcholine to produce PA and choline, and this function is dependent on PLD2 catalytic domain HKD. NFOT is an allosteric and catalytic inhibitor, targets two different sites in HKD and PH domain, suppressing both PA production and PIP2 binding to PLD2. FIPI is specific catalytic inhibitor to PLD and binds to HKD domain, inhibiting PA production (Monovich et al., 2007). The alcohol 1-butanol also blocks PLD-catalyzed PA generation (McDermott, Wakelam & Morris, 2004). In order to clarify whether PLD2 catalytic activity was involved in spindle migration, oocytes were treated with two catalytic inhibitors, FIPI and 1-butanol, in the same manner of NFOT. Beyond our expectation, we were astonished to find that FIPI nearly exerted no effect on spindle migration even in a cell-death dose (Fig. 4). Similarly, 0.1% 1-butanol did not disrupt the spindle movement but indeed enlarged the size of spindle (Fig. 5). There data inferred that PLD2 HKD domains probably do not take part in the regulation of spindle cortex-towards migration.

PLD2 suppression altered actin dynamics in mouse oocytes

As the spindle is disabled in movement under the conditions of the PLD2 inhibition with NFOT, we went back to assay to see if any changes occurred in the actin network and the relative several proteins, such as PIP2 and Colifin. With the specific method of actin fixation, we found that actin was presented in a fibro-crossing manner throughout the cytoplasm in control oocytes; however, in the NFOT treatment group, actin distribution changed to forming many clusters quite big in size on the oocyte surface, with no obvious actin fiber crossover in cytoplasm (Fig. 6). It is plausible that PLD2 inhibition by NFOT resulted in great change in oocyte actin distribution, which led to the disable movement of spindle from accentor to the cortex.

Since PIP 2 and Colifin are tightly associated with actin polymerization and dynamics, we continued to examine the cellular level of these proteins in NFOT-treated oocytes. By immunofluorescence in normal MI oocytes, we found that both PIP2 and phosphorylated Colifin (p-Colifin^Ser3) were aggregated as bright foci at the poles of spindles (Fig. 7A, 2; Fig. 7B, 3: arrows) and also randomly in cytoplasm area (Fig. 7A, 2; Fig. 7B, 3: arrowheads), just like the signal of microtubule orgalizing center (MTOC); however, the foci signal of PIP2 definitely disappeared in oocytes incubated with NFOT (Fig. 7A, 6); similarly, p-Colifin^Ser3 signal was also significantly weakened or totally disappeared in NFOT-treated oocytes (Fig. 7B, 7). Further statistical analysis confirmed that the number of oocytes with bright foci of PIP2 or p-Colifin^Ser3 was significantly reduced after NFOT treatment (Figs. 7C and 7D) (P < 0.001). Consistent with this Western blot analysis indicated that the p-Colifin^Ser3 protein level was reduced greatly while the PLD2 level remained stable (Fig. 7E), and this was supported by statistical data that the band gray intensity of p-Cofilin^Ser3 was significantly decreased in NFOT-treated oocytes (Fig. 7F). The two weakened signals indirectly indicate that at least at the spindle poles the actin was more likely to be in a disassembly state under the condition of PLD2 inhibition, which may stand for the unmoved spindles.

In addition, we found the small GTPase RhoA, which can bind with PLD2 and regulate actin dynamics, was organized into a bi-polar structure with PLD2 localized mainly at the polar area in MI oocytes (Fig. 8). In NFOT-treated oocytes, RhoA sustained in “spindle-like” assembly, and also concentrated as additional dots in the cytoplasm, which were co-localized with the cytoplasmic PLD2 dots (Fig. 8A, 6–8: arrows). Results of statistical analysis confirmed the proportion of oocytes with cytoplasmic RhoA dots was pronouncedly higher than that in control (Fig. 8B) (P < 0.001). The changed distribution of RhoA must affect its function in regulation of actin polymerization and dynamics. NFOT-induced big dots in the cytoplasm are newly aggregated structure, may have some relationship with some transport-vesicles in the cytoplasm such as Golgi stacks and endosomes.