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Progesterone is widely used to induce maturation of isolated fully grown oocytes of the African clawed frog, Xenopus laevis. However, the hormone fails to release oocytes from the layer of surrounding follicle cells. Here, we report that maturation and follicle rupture can be recapitulated in vitro by treating isolated follicular oocytes with progesterone and low doses of the matrix metalloproteinase (MMP), collagenase, which are ineffective in the absence of the steroid. Using this in vitro ovulation model, we demonstrate that germinal vesicle breakdown (GVBD) and oocyte liberation from ovarian follicles occur synchronously during ovulation. Inhibition of the MAPK pathway in these experimental settings suppresses both GVBD and follicular rupture, whereas inhibition of MMP activity delays follicular rupture without affecting GVBD. These results highlight importance of MAPK and MMP activities in the ovulation process and provide the first evidence for their involvement in the release of oocytes from ovarian follicles in frogs. The in vitro ovulation model developed in our study can be employed for further dissection of ovulation.
Figure 1. Effects of progesterone (PG) and human chorionic gonadotropin (hCG) on Xenopus follicular oocytes in vitro. (A) Morphology of isolated stage VI follicles before and after hormonal treatment. The white arrowheads in the panel point to the thin blood capillaries visible in the follicle layer. (B) Time course of germinal vesicle breakdown (GVBD) and follicle rupture in the hormone-treated oocytes. (C) Effects of the ovulation hormones on defolliculated oocytes. To remove the follicle layer, oocytes were treated with 5 mg/mL collagenase for 3 h before hormone administration. The experiment was repeated with five batches of follicular oocytes obtained from different animals. The results of a single batch experiment are presented in the figure.
Figure 2. Effects of PG and hCG on the follicular oocytes in the presence of low concentrations of collagenase. (A) Morphology of isolated follicles before and after hormonal treatment in the presence of 50 µg/mL collagenase. A white arrowhead in the panel points to a remnant of the follicle layer that is still attached to the oocyte after follicle rupture. (B,C) Time courses of GVBD and follicle rupture, correspondingly. The experiment was repeated with five batches of follicular oocytes obtained from different animals, and the results of a single batch experiment are presented.
Figure 3. Effects of PG on the follicular oocytes pretreated with collagenase. Isolated follicles were pretreated with 5 mg/mL collagenase for 30 min before PG administration. (A) Time courses of GVBD and follicle rupture in the PG-treated oocytes; (B) time courses of spontaneous hormone-independent defolliculation in the follicles pretreated with collagenase for 30 min (blue color) or 120 min (red color); (C) time dependency of defolliculation in the presence of 5 mg/mL collagenase. The experiment was repeated with five batches of follicular oocytes obtained from different animals, and the results of a single batch experiment are presented.
Figure 4. Synchronization of GVBD and follicular rupture during in vitro ovulation. (A) Two possible sequences of events during ovulation. The four distinctive morphological types can be observed during this process: oocytes without a white spot surrounded by follicle cells (WS−FL+), oocytes with a white spot and follicle cells (WS+FL+), oocytes without a white spot and follicle cells (WS−FL−), and oocytes with a white spot and without follicle cells (WS+FL−). (B) Ratios of the oocyte phenotypes in the PG-treated follicular culture preincubated for 30 min with 5 mg/mL collagenase; (C) The phenotype ratios in the follicle culture treated with the hormone in the presence of 50 µg/mL of collagenase. (D) Relative frequencies of the two intermediate phenotypes WS+FL+ and WS−FL− under the conditions described for panels (B,C). The experiment was repeated with five batches of follicular oocytes obtained from different animals, and the results of a single batch experiment are shown in panels (B,C). Bars in panel (D) represent SD values of the mean obtained in five experiments.
Figure 5. Effect of matrix metalloproteinase (MMP) inhibition on oocyte maturation and defolliculation. (A,B) Ratios of the specific oocyte phenotypes, as designated in the legend to Figure 4, in the follicular culture preincubated for 30 min with 5 mg/mL collagenase and treated with PG in the absence or presence of an MMP inhibitor, respectively. The inhibitor was added to the follicular oocytes at a final concentration of 50 µM at the time of PG administration. (C) Relative frequencies of the two intermediate phenotypes, WS+FL+ and WS−FL−, under the conditions described for panels (A,B). Bars in panel (C) represent SD values of the mean obtained in four experiments. An asterisk in panel C indicates statistical significance in the phenotype ratio between A and B (p < 0.05).
Figure 6. Effect of MAPK pathway inhibition on maturation and defolliculation. (A) Phosphorylation status of MAPK in the absence or presence of an MAPKK inhibitor during in vitro ovulation of Xenopus oocytes. The MAPKK inhibitor U0126 was added to the follicular oocytes at a final concentration of 50 µM at the time of PG administration. (B,C) Time courses of GVBD and follicular rupture, respectively. The experiment was repeated with four batches of follicular oocytes obtained from different animals and the results of a single batch experiment are presented.
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