MPF governs the assembly and contraction of actomyosin rings by activating RhoA and MAPK during chemical-induced cytokinesis of goat oocytes

Abstract

The interplay between maturation-promoting factor (MPF), mitogen-activated protein kinase (MAPK) and Rho GTPase during actin-myosin interactions has yet to be determined. The mechanism by which microtubule disrupters induce the formation of ooplasmic protrusion during chemical-assisted enucleation of mammalian oocytes is unknown. Moreover, a suitable model is urgently needed for the study of cytokinesis. We have established a model of chemical-induced cytokinesis and have studied the signaling events leading to cytokinesis using this model. The results suggested that microtubule inhibitors activated MPF, which induced actomyosin assembly (formation of ooplasmic protrusion) by activating RhoA and thus MAPK. While MAPK controlled actin recruitment on its own, MPF promoted myosin enrichment by activating RhoA and MAPK. A further chemical treatment of oocytes with protrusions induced constriction of the actomyosin ring by inactivating MPF while activating RhoA. In conclusion, the present data suggested that the assembly and contraction of the actomyosin ring were two separable steps: while an increase in MPF activity promoted the assembly through RhoA-mediated activation of MAPK, a decrease in MPF activity triggered contraction of the ring by activating RhoA.

Figure 1. Photographs of goat oocytes after different treatments to induce ooplasmic protrusions.

A and A' are the same oocyte observed under phase contrast and fluorescence microscope, respectively, after Hoechst staining. B to K are confocal micrographs, of which, B to E and H to K are merged images with DNA colored blue, α-tubulin green and actin red while F and G with DNA colored red while the activated MAD2 green. A and B show oocytes with protrusions while C shows an oocyte without protrusion after demecolcine treatment. D and E are oocytes with protrusions after treatment with MG132 and caffeine, respectively. F and G are oocytes MAD2-positive and -negative after demecolcine and MG132 treatment, respectively. H and I show D+U- and M+U-treated oocytes with spindles disassembled and intact, respectively. J and K are OA.5h oocytes without protrusion and OA4h oocytes with protrusion, respectively, both showing actin enrichment. Arrow: intact or disintegrated spindles; *: Pb1. The scale bar is 30-µm in A–E and H–K but 70-µm in F and G.

Figure 2. Relative MPF and MAPK activities (A to D) and RhoA-GTP levels (E) in oocytes after different treatments.

In figures A to D, black and gray bars represent MPF and MAPK activities, respectively. MII: Freshly matured oocytes; D and D-: Oocytes with and without protrusion respectively after demecolcine; AD-: Aged oocytes without protrusion after demecolcine; +: In the presence of; R: ROS; U: U0126; Y: Y27632; OA.5h and OA4h: OA for 0.5 and 4 h respectively; MD2: MAD2. a–d: Values without a common letter above their bars differ (P<0.05) within enzyme activities.

Figure 3. Confocal micrographs of oocytes showing enrichment of actin and/or myosin (arrow) after different treatments.

Myosin is shown in green, actin in red and DNA in blue. Treatments include demecolcine (D), MG132 (M) or OA for 0.5 h (OA.5h) alone or demecolcine + Blebbistatin (Bleb), Cytochalasin B (CB), U0126 (U) or ROS (R). *: Pb1. Scale bar is 30 µm.

Figure 4. Ooplasmic protrusion and MPF/MAPK activities after oocytes were injected with mouse sperm head (SP) and treated with demecolcine or MG132.

A and A' are the same oocyte observed under phase contrast and fluorescent microscopes, respectively, showing the egg chromosome-induced protrusion (arrow), SP-induced protrusion (arrowhead) and Pb1 (*). B to E show confocal images with DNA colored blue, α-tubulin green and actin red. B and C: Oocytes with both egg chromosome- and SP-induced protrusions after demecolcine and MG132 treatment, respectively. D and E: Enucleated oocytes injected with SP formed protrusion after MG132 (D) but did not after demecolcine (E). Scale bar is 20 µm. F: Relative MPF/MAPK activities after intact (MII) or enucleated oocytes (EnMII) were injected with SP (+SP) and treated with demecolcine (+SP+D) or MG132 (+M). Values without a common letter above their bars differ (P<0.05) within kinase activities.

Figure 5. Pb2 Extrusion and activities of MPF, MAPK and RhoA after oocytes with demecolcine-induced protrusions (DIP) were activated.

A, A' and D, D' are the same oocyte observed under phase contrast and fluorescence microscope, respectively, after Hoechst staining. B, C, E and F are confocal images with DNA colored blue, α-tubulin green and actin red. Contraction of the actomyosin ring began at 0.5 h (B) and 1 h (E) while Pb2 extrusion (arrows) observed at 1 h (A, A', C) and 2 h (D, D', F) after chemical activation (CA) of the DIP and control MII oocytes, respectively. *: Pb1. Scale bar is 30 µm. Panels G and H show relative MPF/MAPK and RhoA activities, respectively. MII and D: Freshly matured and DIP oocytes prior to CA; +: In the presence of; Y: Y27632; CA.5h, CA1h and CA2h: Oocytes assayed at 0.5, 1 and 2 h of CA, respectively. a–d: Values without a common letter above their bars differ (P<0.05) within enzyme activities.

Figure 6. The signaling pathways leading to the assembly and contraction of actomyosin rings during chemical-induced oocyte cytokinesis.

Refer to the text for detailed explanations.