LIMK1/2 inhibitor LIMKi 3 suppresses porcine oocyte maturation

Antibodies and chemicals

Basic maturation culture medium was TCM 199 (Sigma, St. Louis, MO, USA). A rabbit polyclonal p-LIMK-1/2 antibody was from Santa Cruz (Santa Cruz, CA, USA). An Alexa Fluor 488 secondary antibody was from Invitrogen (Carlsbad, CA, USA). Phalloidin-TRITC and mouse monoclonal anti-α-tubulin-FITC antibody were from Sigma. LIMK inhibitor LIMKi 3 was from Calbiochem (Darmstadt, Germany).

Oocyte collection and culture

Animal use was conducted in accordance with the Animal Research Institute Committee guidelines of Nanjing Agricultural University, China. This study was specifically approved by the Committee of Animal Research Institute, Nanjing Agricultural University, China. Porcine ovaries were collected from a local slaughterhouse, and were then transported to laboratory within 3 h in sterile saline (0.9% NaCl) containing 500 IU/mL of penicillin and 500 IU/mL of streptomycin at 37 °C. Cumulus-oocyte complexes (COCs) were obtained from medium-sized follicles of ovaries by aspirating with a 20-gauge needle attached to a 5-ml disposable syringe. Oocytes surrounded by a compact cumulus mass and also with uniform ooplasm were selected and transferred to Dulbecco’ s PBS, then washed three times with modified medium 199 containing 0.1% (wt/vol) polyvinyl alcohol, 0.91 mM sodium pyruvate, 3.05 mM glucose, 75 mg/L of penicillin, and 50 mg/L of streptomycin. Only oocytes with intact cumulus cells and evenly granulated ooplasm were chosen for in vitro maturation (IVM).

A group of 80 COCs were transferred to 500 µL of maturation medium consisting of 90% (vol/vol) modified M199, 10 ng/mL of EGF (mouse EGF; Sigma), 10 IU/mL of hCG, 10 IU/mL of pregnant mare’s serum gonadotropin (PMSG), 0.57 mM cysteine (Sigma),and 10% (vol/vol) pig follicular fluid, and then covered with 200 µL paraffin oil in a four-well dish (NUNC) for 26 h (for COCs at metaphase I (MI)) or 44 h (for COCs at metaphase II (MII)) at 38.5 °C in a 5% CO₂ atmosphere.

LIMK1/2 inhibitor LIMKi 3 treatment

For LIMK1/2 inhibitor treatment, stock LIMKi 3 (50 mM in dimethylsulfoxide (DMSO)) was diluted in M199 to final concentrations of 50, 100, 150 and 200 µM. A control group was cultured in DMSO at the same relative concentration of solvent. COCs were cultured with LIMKi 3 to evaluate its effects on oocyte maturation. COCs were denuded of their cumulus cells by gentle pipetting with 0.1% (w/v) hyaluronidase (Sigma). Oocytes with clearly extruded polar bodies were considered to be matured. The occurrence of first polar body extrusion in oocytes was examined after removing cumulus cells.

Rescue experiment

After cultured in maturation medium M199 containing 200 µM LIMK inhibitor LIMKi 3 for 44 h, treated oocytes were washed three times in fresh culture solution (2 min each wash). Oocytes were then transferred to fresh culture solution and cultured for an additional 16 h under paraffin oil at 38.5 °C in a 5% CO₂ atmosphere.

Immunofluorescence staining and confocal microscopy

For immunofluorescence staining of LIMK1/2, actin, and α-tubulin, oocytes were fixed in 4% paraformaldehyde in PBS at room temperature for 30 min and then transferred to a membrane permeabilization solution (0.5% Triton X-100) for 8–12 h at room temperature. To suppress nonspecific binding of IgG, oocytes were blocked in blocking buffer for 1 h at room temperature (1% BSA-supplemented PBS). Then oocytes were incubated overnight at 4 °C or 5 h at room temperature with a rabbit polyclonal p-LIMK-1/2 antibody (1:50) or anti-α-tubulin-FITC (1:200) for 3 h or with 2 µg/mL of Phalloidin-TRITC at room temperature for 1 h. After three washes (2 min each) in wash buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS), oocytes were labeled with Alexa Fluor-488 goat anti-rabbit IgG (1:100; for p-LIMK-1/2 staining) for 1 h at room temperature. Oocytes were stained with Hoechst 33342 for 10 min, mounted on glass slides, and then examined with a confocal laser-scanning microscope (Zeiss LSM 700 META; Zeiss, Oberkochen, Germany). Each experiment was repeated three times; at least 30 oocytes were examined per experimental condition.

Fluorescence intensity analysis

To analyze the fluorescence intensity of actin filaments, samples of control and treated oocytes were mounted on the same glass slide. Image J software was used to define a region of interest (ROI), and the average fluorescence intensity per unit area within the ROI was determined. Independent measurements using identically sized ROIs were taken for the cell membrane and cytoplasm. The average values of all measurements were used to determine the final average intensity between control and treated oocytes.

Western blot analysis

A total of 100 porcine oocytes were collected, lysed in Laemmli sample buffer (SDS sample buffer with 2-mercaptoethanol), and boiled at 100 °C for 10 min. Proteins were separated by SDS-PAGE and then electrophoretically transferred to polyvinylidene fluoride membranes. To avoid nonspecific binding, membranes were blocked with Tris-buffered saline (TBS) containing 0.1% (w/w) Tween 20 (TBST) and 5% (w/v) nonfat dry milk powder for 1 h at room temperature. The membranes were simultaneously incubated at overnight 4 °C with a rabbit polyclonal p-LIMK-1/2 (1:200, Santa Cruz, USA) or rabbit monoclonal anti-α-tubulin antibody (1:2,000; Cell Signaling Technology, Danvers, MA, USA). After washing three times in TBST (10 min each), the membranes were incubated for 2 h with secondary anti-rabbit HRP-conjugated antibodies (1:2,000; Cell Signaling Technology, Beverly, MA, USA) in 5% nonfat dry milk in TBST at room temperate. Finally, the membranes were washed 3 times in TBST and then the specific proteins were visualized using chemiluminescence reagent (Millipore, Billerica, MA).

Statistical analysis

A group of 80 oocytes were cultured with or without LIMKi 3 for once experimental condition and at least three replicates were performed for each treatment. Results were expressed as means ± standard errors of the mean. Statistical analysis was conducted using analysis of variance (ANOVA), and differences between treatment groups were assessed by Duncan’s multiple comparison test. P < 0.05 was considered significant.

P-LIMK1/2 expression in porcine oocyte

The subcellular localizations of p-LIMK-1/2 at different stages of meiosis were assessed by immunofluorescent staining. As shown in Fig. 1A, p-LIMK-1/2 was primarily localized at cortex of oocyte during all meiotic stages, and this localization was similar with actin. Oocytes of GV stage were obtained and were cultured for 26 and 44 h, which corresponded to the times to achieve the MI and MII stages, respectively. Using western blot, we found that p-LIMK-1/2 protein was all expressed at the MI and MII stages (Fig. 1B).

LIMKi 3 treatment suppresses polar body extrusion in vitro

Based on the examination of LIMK1/2 expression and localization in porcine oocytes, we next examined whether LIMK1/2 inhibitor LIMKi 3 affected porcine oocyte maturation. As shown in Fig. 2A, COCs exhibited different diffusion situation between control and treatment group: the diffusion of 200 µM LIMKi 3 treated-COCs was significantly weaker than the control group. In the standard of COCs surrounded by the cumulus cell layers, we counted the proportion of diffusion and found that the rate of good diffusion was decreased significantly (40.05 ± 3.03% vs. 20.50 ± 4.05%; p < 0.05). Oocytes were then obtained from COCs after hyaluronidase treatment, we found that few oocytes extruded the polar body after 200 µM LIMKi 3 treatment. In addition, the rate of polar body extrusion of oocytes was reduced in a dose-dependent manner (Fig. 2B). For controls, the ratio of polar body extrusion was 73.44 ± 5.18% (n = 123); while oocytes were treated with LIMKi 3 in the concentration of 50 µM, 100 µM, 150 µM, 200 µM, the ratio decreased to 65.66 ± 3.24% (n = 95; p > 0.05), 58.04 ± 5.58% (n = 111; p < 0.05), 54.76 ± 3.00% (n = 101; p < 0.05), 46.88 ± 5.04% (n = 135; p < 0.01). Therefore, the porcine oocyte maturation was suppressed in the treatment of LIMKi 3, and 200 µM was chosen as the appropriate concentration for subsequent experiments.

To further clarifly the effects of LIMKi 3 on porcine oocyte maturation, we prolonged the culture time of porcine oocyte to 60 h. As shown in Fig. 2C, after prolonging the culture time, the maturation rate increased compared with 44 h culture group (70.68 ± 6.99% vs. 47.61 ± 7.92%). Thus, LIMKi 3 repressed the cell cycle of porcine oocyte maturation. To further confirm the inhibitory effects of LIMKi 3, we did the rescue experiment. Compared with oocytes before rescue (78.18 ± 4.69% vs. 47.61 ± 7.92%; p < 0.01), LIMKi 3 treated oocytes reached the MII stage (200 µM) after an additional 16 h of culture in fresh medium, with the ratio of 73.06 ± 3.09% (Fig. 2D). Therefore, LIMKi 3 may have a repressive effect on porcine oocyte maturation and its effect was reversible.

LIMKi 3 treatment causes disruption of actin distribution in porcine oocyte

Considering that LIMK1/2 has been known to regulate actin cytoskeleton, we next examined the effects of LIMK inhibitor LIMKi 3 on actin filament distribution of meiosis I in porcine oocyte. As shown in Fig. 3A (10X), compared with the control oocytes, actin fluorescence intensity of LIMKi 3 treated oocytes became much weaker. Furthermore, actin distribution was disturbed both at the membrane and cytoplasm of oocyte after LIMKi 3 treatment (Fig. 3A (40X)). The value of actin fluorescence intensity in porcine oocyte was analyzed by Image J software (Fig. 3B). For membrane actin fluorescence intensity, the rate of LIMKi 3 treated oocytes was significantly lower than that in control oocytes (78.88 ± 16.53% vs. 48.18 ± 9.14%; P < 0.01). Compared with control oocytes, the rate of cytoplasmic actin also decreased after LIMKi 3 treatment (29.76 ± 9.02% vs. 22.70 ± 7.81%; P < 0.01). These results indicated that the LIMKi 3 treatment resulted in an abnormal actin distribution.

LIMKi 3 treatment causes abnormity of spindle positioning in porcine oocyte

Spindle migration in meiosis I is actin dependent, so we next investigated the effects of LIMK1/2 inhibitor LIMKi 3 on spindle positioning at MI stage. As shown in Fig. 4A (10X), most of the spindles exhibited at the position distant with the cortex after treatment with 200 µM LIMKi 3, which is different with the spindles of control oocytes that exhibited peripheral localization. Enlarged images more clearly showed the difference between control and treatment (Fig. 4A (40X)).

We also analyzed the cell cycle stage after culture for 26 h. As shown in Fig. 4B, the percentage of oocytes with spindles closed to cytoplasm center at the MI stage for LIMKi 3 treatment was clearly higher than that of control oocytes (57.11 ± 7.85%, n = 212 vs. 26.58 ± 5.18%, n=234) (p < 0.01), nevertheless the percentage of oocytes with spindles at the cortex in the MI stage (35.27 ± 7.94% vs. 59.16 ± 11.19%; p < 0.01) and in the anaphase/telophase (AT) stage (3.27 ± 4.53% vs. 9.61±6.04%; p < 0.05) were reduced after LIMKi 3 treatment. Meanwhile, the percentage of oocytes at the GVBD stage showed no significant difference between control and treatment (4.65 ± 3.08% vs. 4.35 ± 4.00%; p > 0.05). These results demonstrated that LIMKi 3 treatment effectively affected spindle positioning.