Roles of MAPK and spindle assembly checkpoint in spontaneous activation and MIII arrest of rat oocytes

Abstract

Rat oocytes are well known to undergo spontaneous activation (SA) after leaving the oviduct, but the SA is abortive with oocytes being arrested in metaphase III (MIII) instead of forming pronuclei. This study was designed to investigate the mechanism causing SA and MIII arrest. Whereas few oocytes collected from SD rats at 13 h after hCG injection that showed 100% of mitogen-activated protein kinase (MAPK) activities activated spontaneously, all oocytes recovered 19 h post hCG with MAPK decreased to below 75% underwent SA during in vitro culture. During SA, MAPK first declined to below 45% and then increased again to 80%; the maturation-promoting factor (MPF) activity fluctuated similarly but always began to change ahead of the MAPK activity. In SA oocytes with 75% of MAPK activities, microtubules were disturbed with irregularly pulled chromosomes dispersed over the spindle and the spindle assembly checkpoint (SAC) was activated. When MAPK decreased to 45%, the spindle disintegrated and chromosomes surrounded by microtubules were scattered in the ooplasm. SA oocytes entered MIII and formed several spindle-like structures by 6 h of culture when the MAPK activity re-increased to above 80%. While SA oocytes showed one Ca(2+) rise, Sr(2+)-activated oocytes showed several. Together, the results suggested that SA stimuli triggered SA in rat oocytes by inducing a premature MAPK inactivation, which led to disturbance of spindle microtubules. The microtubule disturbance impaired pulling of chromosomes to the spindle poles, caused spindle disintegration and activated SAC. The increased SAC activity reactivated MPF and thus MAPK, leading to MIII arrest.

Figure 1. A diagram depicting the overall hypothesis of the study.

Refer to the last paragraph of the Introduction section for detailed explanations.

Figure 2. Confocal images of rat oocytes at different stages of IA (left column) or SA (right column).

The DNA and α-tubulin in oocytes were pseudo-colored blue and green, respectively. A is an oocyte at the metaphase II (MII) stage showing a regular spindle with chromosomes aligned on the metaphase plate. B is an IA oocyte in anaphase II (AnII) showing a spindle with chromosomes tidily aligned on either pole. C is an IA oocyte in early telophase II (e-TelII) with a condensed chromosome mass on either pole of the spindle and the initiation of PB2 extrusion. D is an IA oocyte in late telophase II (l-TelII) with extruded PB2 and the initiation of chromosome decondensation. E is an IA oocyte in interphase (Int) with pronuclear formation. F is a SA oocyte in AnII with chromosomes dispersed over the surface of the spindle. G is a SA oocyte in e-TelII with chromosomes arranged toward the spindle poles. H and I are SA oocytes in l-TelII showing disintegrated spindles with chromosomes surrounded by microtubules scattered in the ooplasm. J is a SA oocyte at the MIII stage with microtubules reorganized into several small spindles around the scattered chromosomes. PB2 (arrow) was often observed in IA oocytes but not in SA oocytes. Scale bar is 20 µm.

Figure 3. Changes in MPF and MAPK activities of rat oocytes during SA of in vitro culture or during IA after Sr²⁺ treatment.

Oocytes for SA were collected 19 h post hCG and cultured for different times in mR1ECM medium before kinase assay. For IA, newly ovulated oocytes collected 13 h post hCG injection were treated with SrCl₂ for 15 min and were assayed for kinase activities at different times after SrCl₂ treatment. a-g: Values without a common letter differ (P<0.05).

Figure 4. Distribution and quantification of phosphorylated MAPK (p-MAPK) in rat oocytes.

Oocytes collected 19 h post hCG were aged for different times in mR1ECM before examination for p-MAPK expression. A. Laser confocal micrographs showing p-MAPK distribution. Whereas images in different rows show a non-SA oocyte remaining at the MII stage (nSA), a SA oocyte observed at 1 h (SA1h) and a SA oocyte observed at 6 h (SA6h) of in vitro aging, respectively, images in different columns show α-tubulin, p-MAPK, chromatin and merged pictures, respectively. The scale bar is 20 µm. B is a graph showing p-MAPK quantification in SA and non-SA (NSA) oocytes aged in vitro for 0, 1 and 6 h. Each treatment was repeated 3 times and each replicate contained 6–8 oocytes. Values without a common letter above their bars differ (p<0.05).

Figure 5. Laser confocal micrographs showing BUB1 in rat oocytes.

Oocytes collected 19 h post hCG were aged for different times in mR1ECM before examination for BUB1. The α-tubulin, BUB1 and chromatin were pseudo-colored green, red and blue, respectively. Images in different rows show freshly collected oocyte in MII (FCO), non-SA oocytes observed at 0.5 h (NSA0.5h) and 6 h (NSA6h), and SA oocytes observed at 0.5 h (SA0.5h), 4 h (SA4h) and 6 h (SA6h) of in vitro aging, respectively. Images in different columns show α-tubulin, BUB1, chromatin and merged pictures, respectively. Scale bars are 15 µm.

Figure 6. Ca²⁺ oscillations during IA or SA of rat oocytes.

Figure 7. Possible pathways leading to the MIII arrest in SA oocytes and the pronuclear formation in IA oocytes.

For a detailed explanation, please refer to the text in the Discussion section. Because APC activity was not actually examined in this study, dotted lines were used to depict the possible pathways involving the APC activity in this figure.