Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
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.
Baulieu,
Steroid-induced meiotic division in Xenopus laevis oocytes: surface and calcium.
1978, Pubmed,
Xenbase
Baulieu,
Steroid-induced meiotic division in Xenopus laevis oocytes: surface and calcium.
1978,
Pubmed
,
Xenbase
Bayaa,
The classical progesterone receptor mediates Xenopus oocyte maturation through a nongenomic mechanism.
2000,
Pubmed
,
Xenbase
Borman,
Progesterone promotes oocyte maturation, but not ovulation, in nonhuman primate follicles without a gonadotropin surge.
2004,
Pubmed
Brännström,
Progesterone is a mediator in the ovulatory process of the in vitro-perfused rat ovary.
1989,
Pubmed
Cameron,
Activation of mitogen-activated protein kinases by gonadotropins and cyclic adenosine 5'-monophosphates in porcine granulosa cells.
1996,
Pubmed
Castro,
Cyclin B/cdc2 induces c-Mos stability by direct phosphorylation in Xenopus oocytes.
2001,
Pubmed
,
Xenbase
Chaffin,
Local role of progesterone in the ovary during the periovulatory interval.
2002,
Pubmed
Curry,
Impact of extracellular matrix remodeling on ovulation and the folliculo-luteal transition.
2006,
Pubmed
DETTLAFF,
THE R OLE OF THE GERMINAL VESICLE IN OOECYTE MATURATION IN ANURANS AS REVEALED BY THE REMOVAL AND TRANSPLANTATION OF NUCLEI.
1964,
Pubmed
Deng,
Nongenomic steroid-triggered oocyte maturation: of mice and frogs.
2009,
Pubmed
,
Xenbase
Dumont,
Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals.
1972,
Pubmed
,
Xenbase
Espey,
Effects of epostane on ovarian levels of progesterone, 17 beta-estradiol, prostaglandin E2, and prostaglandin F2 alpha during ovulation in the gonadotropin-primed immature rat.
1990,
Pubmed
Fan,
MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility.
2009,
Pubmed
Ferrell,
Building a cellular switch: more lessons from a good egg.
1999,
Pubmed
,
Xenbase
Fortune,
Steroid production by Xenopus ovarian follicles at different developmental stages.
1983,
Pubmed
,
Xenbase
Fortune,
Production of androgen and estradiol-17 beta by Xenopus ovaries treated with gonadotropins in vitro.
1981,
Pubmed
,
Xenbase
Haccard,
Naturally occurring steroids in Xenopus oocyte during meiotic maturation. Unexpected presence and role of steroid sulfates.
2012,
Pubmed
,
Xenbase
Hammes,
Steroids and oocyte maturation--a new look at an old story.
2004,
Pubmed
,
Xenbase
Hibbert,
Midcycle administration of a progesterone synthesis inhibitor prevents ovulation in primates.
1996,
Pubmed
Hsieh,
Genetic dissection of epidermal growth factor receptor signaling during luteinizing hormone-induced oocyte maturation.
2011,
Pubmed
Jacobelli,
Hormone-induced maturation of Xenopus laevis oocytes: effects of different steroids and study of the properties of a progesterone receptor.
1974,
Pubmed
,
Xenbase
Kosako,
Requirement for the MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation.
1994,
Pubmed
,
Xenbase
Liu,
Transcription-dependent and transcription-independent functions of the classical progesterone receptor in Xenopus ovaries.
2005,
Pubmed
,
Xenbase
Lutz,
Evidence that androgens are the primary steroids produced by Xenopus laevis ovaries and may signal through the classical androgen receptor to promote oocyte maturation.
2001,
Pubmed
,
Xenbase
Lydon,
Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities.
1995,
Pubmed
Masui,
Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes.
1971,
Pubmed
Masui,
Oocyte maturation.
1979,
Pubmed
Ogiwara,
Gelatinase A and membrane-type matrix metalloproteinases 1 and 2 are responsible for follicle rupture during ovulation in the medaka.
2005,
Pubmed
Ogiwara,
A new in vitro ovulation model for medaka based on whole ovary culture.
2010,
Pubmed
Palmer,
A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1.
1998,
Pubmed
,
Xenbase
Panigone,
Luteinizing hormone signaling in preovulatory follicles involves early activation of the epidermal growth factor receptor pathway.
2008,
Pubmed
Peter,
A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK.
2002,
Pubmed
,
Xenbase
Reynhout,
Response of large oocytes of Xenopus laevis to progesterone in vitro in relation to oocyte size and time after previous HCG-induced ovulation.
1975,
Pubmed
,
Xenbase
Robker,
Ovulation: a multi-gene, multi-step process.
2000,
Pubmed
Robker,
Progesterone-regulated genes in the ovulation process: ADAMTS-1 and cathepsin L proteases.
2000,
Pubmed
Schuetz,
Evidence for follicle wall involvement in ovulation and progesterone production by frog (Rana pipiens) follicles in vitro.
1982,
Pubmed
Skory,
Engineering the ovarian cycle using in vitro follicle culture.
2015,
Pubmed
Smith,
In vitro induction of physiological maturation in Rana pipiens oocytes removed from their ovarian follicles.
1968,
Pubmed
Smith,
The interaction of steroids with Rana pipiens Oocytes in the induction of maturation.
1971,
Pubmed
Snyder,
Inhibition of ovulation in rats with epostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase.
1984,
Pubmed
Su,
Oocyte-dependent activation of mitogen-activated protein kinase (ERK1/2) in cumulus cells is required for the maturation of the mouse oocyte-cumulus cell complex.
2003,
Pubmed
Subtelny,
Maturation of ovarian frog eggs without ovulation.
1968,
Pubmed
Tajima,
Down-regulation of steroidogenic response to gonadotropins in human and rat preovulatory granulosa cells involves mitogen-activated protein kinase activation and modulation of DAX-1 and steroidogenic factor-1.
2003,
Pubmed
Takahashi,
Recent advances in the understanding of teleost medaka ovulation: the roles of proteases and prostaglandins.
2013,
Pubmed
Tokmakov,
Unfertilized frog eggs die by apoptosis following meiotic exit.
2011,
Pubmed
,
Xenbase
Tokmakov,
Inhibition of MAPK pathway by a synthetic peptide corresponding to the activation segment of MAPK.
1998,
Pubmed
,
Xenbase
WRIGHT,
Influence of estrogens on induction of ovulation in vitro in Rana pipiens.
1961,
Pubmed
Yang,
Xenopus laevis ovarian CYP17 is a highly potent enzyme expressed exclusively in oocytes. Evidence that oocytes play a critical role in Xenopus ovarian androgen production.
2003,
Pubmed
,
Xenbase
el-Zein,
The dynamics of the steroidogenic response of perifused Xenopus ovarian explants to gonadotropins.
1988,
Pubmed
,
Xenbase
