Cumulus cells secrete a meiosi‐inducing substance by stimulation with forskolin and dibutyric cyclic adenosine monophosphate

Mitogen-activated protein (MAP) kinase has been reported to be activated during oocyte meiotic maturation in a variety of mammalian species. However, the mechanism(s) responsible for MAP kinase activation and the consequence of its premature activation during gonadotropin-induced oocyte meiotic resumption have not been examined. The present experiments were conducted to investigate the possible role of MAP kinase in FSH-induced and spontaneous oocyte meiotic resumption in the mouse. MAP kinase kinase (MAPKK, MEK) inhibitor, PD98059 or U0126, produced a dose-dependent inhibitory effect on both FSH-induced oocyte meiotic resumption and MAP kinase activation in the oocytes. However, the same inhibitor did not block spontaneous meiotic resumption of either denuded or cumulus cell-enclosed mouse oocytes, despite the activity of MAP kinase being totally inhibited. Immunoblotting the oocytes and the cumulus cells with the anti-active MAP kinase antibody showed that MAP kinase activity in the oocytes was detected at 8 h of FSH treatment, prior to germinal vesicle breakdown and increased as maturation progressed in the following culture period. In the cumulus cells, MAP kinase was activated even faster, its activity was detected at 1 h of FSH stimulation and increased gradually until 8 h of FSH treatment, then decreased and diminished after 12 h of FSH action. These data demonstrated that the MEK-MAP kinase pathway is implicated in FSH-induced but not spontaneous oocyte meiotic resumption.

Section: Discussionmentioning

confidence: 93%

Involvement of MEK-Mitogen-Activated Protein Kinase Pathway in Follicle-Stimulating Hormone-Induced but Not Spontaneous Meiotic Resumption of Mouse Oocytes1

Rubinstein

Luria

et al. 2001

“…In the absence of FSH, 20%-30% of milrinone-treated oocytes remain arrested at the GV stage for up to 48 h of culture, but addition of FSH, while initially delaying GVBD, eventually induces all milrinonetreated oocytes to undergo GVBD by 24 h. This observation may be the first model described in a ruminant species for induced oocyte maturation (as opposed to spontaneous maturation, in which simple removal of oocytes from their follicle results in meiotic resumption [44]). In vivo, meiosis resumes in response to the preovulatory surge of gonadotropins-the mechanism by which this occurs is not clear; however, several studies have suggested production of a granulosa/CC-derived positive stimulus in response to the gonadotropic surge that actively promotes meiotic resumption [45][46][47]. In line with the results of the current study, Byskov et al [48] demonstrated that hypoxanthine-arrested mouse COCs secrete a meiosis-activating substance following FSH, but not LH or hCG stimulation, that could override the effects of the meiotic inhibitor.…”

Section: Discussionmentioning

confidence: 98%

Effect of Specific Phosphodiesterase Isoenzyme Inhibitors During In Vitro Maturation of Bovine Oocytes on Meiotic and Developmental Capacity1

Thomas

Thompson

Armstrong

et al. 2004

120

102

Compared with oocytes matured in vivo, in vitro-matured oocytes are compromised in their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3- (oocyte) and isoenzyme 4- (granulosa cell) specific PDE inhibitors on the kinetics of in vitro maturation and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 microM). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9 h, but they completely blocked meiotic resumption during this period (P < 0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% at germinal vesicle stage after 48 h) was overridden by 24 h when treated with FSH, but not with hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 PDE inhibitor significantly (P < 0.05) increased embryo development to the blastocyst stage by 33%-39% (to an average of 52% blastocysts) compared with control oocytes (38%) after insemination at 28 h, and significantly (P < 0.05) increased blastocyst cell numbers when inseminated at 24 h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the CCs and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential.

“…Culture of mouse isolated oocytes with hypoxanthine, an inhibitor of the spontaneous resumption of meiosis, suggested that a positive regulator produced by somatic cells is transported to the oocyte and induces its maturation [3][4][5][6]. Using the same model system, Byskov et al [7] identified this activity as a sterol.…”

Section: Introductionmentioning

confidence: 92%

Meiosis-Activating Sterol Synthesis in Rat Preovulatory Follicle: Is It Involved in Resumption of Meiosis?1

Cao

Pomerantz

Popliker

et al. 2004

Meiosis-activating sterol (MAS) was shown to overcome the inhibitory effect of hypoxanthine on spontaneous maturation of mouse oocytes and was suggested to mediate the stimulation of meiosis by gonadotropins. Follicular fluid (FF)-MAS is synthesized by cytochrome P450 lanosterol 14alpha-demethylase (LDM). Follicular LDM was preferentially localized in oocytes by immunohistochemistry. Using [3H]acetate or R-[5-3H]mevalonate as precursors as well as high-performance liquid chromatographic and thin-layer chromatographic separation, we have measured the concentrations of de novo-synthesized lanosterol, FF-MAS, and cholesterol in rat graafian follicles, cumulus-oocyte complexes (COCs), and denuded oocytes (DOs) treated with LH, AY-9944 (an inhibitor of Delta14-reductase, which was anticipated to increase FF-MAS levels by inhibiting its metabolism), or both after 8 h of culture. In follicles, both LH and AY-9944 increased the accumulation of FF-MAS as compared to controls. In COCs, AY-9944 caused a marked increase in FF-MAS, but we were unable to detect accumulation of FF-MAS in DOs. Neither the endogenous increases in FF-MAS accumulation nor the addition of FF-MAS to the culture medium could overcome the inhibition on resumption of meiosis by phosphodiesterase inhibitors. Compared to LH-induced resumption of meiosis in follicles, that induced by AY-9944 was much delayed. These results call into question any role of FF-MAS as an obligatory mediator of LH activity on germinal vesicle breakdown. The discrepancy between the positive staining for LDM in oocytes and our inability to detect de novo synthesized FF-MAS in DOs may relate to the sensitivity of the methodology employed and either the number of oocytes used or a deficiency in LDM synthetic activity in such oocytes. Further studies are required to confirm any of these alternatives.