Sustained activity of the EGF receptor is an absolute requisite for LH-induced oocyte maturation and cumulus expansion

Abstract

Mammalian reproduction depends on the release of a mature oocyte from the ovarian follicle. Maturation of the oocyte and rupture of the follicle wall constitute part of the responses to the preovulatory surge of LH, which also include cumulus expansion and granulosa cell luteinization. It was previously shown that the epidermal growth factor receptor (EGFR) mediates the ovulatory response to LH in the ovarian follicle. We hypothesized that it is a sustained activity of the EGFR that generates oocyte maturation and cumulus expansion. We demonstrated that, whereas a transient exposure of rat isolated, intact, preovulatory follicles to either LH or forskolin was sufficient to induce oocyte maturation and cumulus expansion, these LH-induced responses were only generated upon a prolonged activity of the EGFR. In addition, the continuous activity of the EGFR is essential for the chronic phosphorylation of the ERK1/2 downstream signaling molecules, which were shown to be essential for oocyte maturation and cumulus expansion. Interestingly, EGFR-sustained activity was also necessary to maintain the up-regulation of Ptgs2, a gene essential for cumulus expansion. The unusual prolonged duration of ERK1/2 activity may possibly be attributed to the late induction of the ERK-specific phosphatase 3, demonstrated herein. These new data shed light on the unique characteristics of EGFR-ERK1/2 activity in the ovarian follicle and emphasize the fact that the ovulatory process involves a nonclassical activation of this pathway.

Fig. 1.

Transient exposure to LH as well as a brief activation of adenylyl cyclase by forskolin induce oocyte maturation and cumulus expansion. A and B, Isolated intact ovarian follicles were exposed to LH for 20 min, after which time they were washed and placed in a hormone-free medium for a total incubation period of 10 h. As positive and negative controls, ovarian follicles were exposed to LH or vehicle for 10 h. C and D, A similar experiment was performed with forskolin (FSK), a reversible adenylyl cyclase activator. Ovarian follicles were exposed to FSK for 4 and 20 min, after which time they were washed and placed in FSK-free medium for 10 h. Other groups of follicles were exposed to either FSK or vehicle for 10 h. At the end of the incubation period, the follicles were incised. The COCs were monitored for oocyte maturation, as indicated by GVB (A and C) and for the extent of the cumulus expansion. The fraction of expanded, out of the total cumuli examined, is presented in panels B and D. The histograms show means ± se of three independent experiments. Columns with different superscripts differ significantly (P < 0.005 in A–C; P < 0.05 in D).

Fig. 2.

A sustained activity of the EGFR is necessary to mediate the LH-induced oocyte maturation and cumulus expansion. Isolated intact preovulatory follicles were incubated with LH for 10 h, and the EGFR antagonist AG1478 (AG) was added at different time points. As a positive control, AG1478 was not added to the incubation medium and as negative controls, ovarian follicles were incubated without LH or preincubated with AG1478 for 1 h before the addition of LH (lane 2). At the end of the incubation period, the follicles were incised, and the COCs were monitored for oocyte maturation, as indicated by GVB (A) and for the extent of cumulus expansion (B). The histograms show means ± se of at least three independent experiments. Columns with different superscripts differ significantly (P < 0.05).

Fig. 3.

A Sustained activity of the EGFR is required for the LH-induced elevation of Ptgs2 mRNA expression. Isolated intact preovulatory follicles were exposed to LH for 2 h, at which time either AG1478 or vehicle was added for 15, 30, and 60 min. At the end of incubation, the follicles were transferred into liquid nitrogen, their RNA was extracted, and cDNA was then synthesized and subjected to Q-PCR. The graph shows means ± se of three independent experiments, *, P < 0.05 vs. corresponding LH treatment.

Fig. 4.

A sustained activity of the EGFR induces prolonged ERK1/2 phosphorylation. A, Preovulatory follicles were incubated with LH for different time intervals, and AG1478 or vehicle was added 15 min before the end of the incubation period. As a positive control, a group of follicles was treated only with LH (lane 3) and as negative controls, ovarian follicles were incubated without LH (lane 1) or preincubated with AG1478 for 1 h before the addition of LH (lane 2). The follicles were then lysed, and the extracted proteins were subjected to SDS-PAGE, followed by Western blot analysis using antibodies recognizing the phosphorylated, active ERK1/2 (pERK1/2) and those raised against general ERK1/2 (gERK1/2). B, Densitometric quantification of the results normalized against gERK1/2 protein levels. For quantification, intensity values of the bands were measured from three different repeats using image J program. The histograms show means ± se of three independent experiments (*, P < 0.05 vs. corresponding LH treatment).

Fig. 5.

The chronic EGFR activity maintains ERK1/2 in its phosphorylated state. A, Preovulatory follicles were incubated with LH for 1 h, at which time AG1478 or vehicle was added for 15, 30, and 90 min. At the end of the incubation period, the follicles were frozen. As a positive control, AG1478 was not added to the incubation medium and as negative controls, ovarian follicles were incubated without LH or preincubated with AG1478 for 1 h before the addition of LH (lane 2). B, Densitometric quantification of the results normalized against gERK1/2 protein levels. The histograms show means ± se of three independent experiments (*, P < 0.05 vs. corresponding LH treatment).

Fig. 6.

A sustained activity of ERK1/2 is essential for the LH-induced oocyte maturation and cumulus expansion. Ovarian follicles were incubated with LH and UO126, the inhibitor of MEK, was added at different time points. As a positive control, UO126 was not added to the incubation medium, and as negative controls, ovarian follicles were incubated without LH or preincubated with UO126 for 1 h before the addition of LH (lane 2). After 10 h of incubation, the follicles were incised and the COCs were monitored for oocyte maturation, as indicated by GVB (A) and for the extent of cumulus expansion (B). The histograms show means ± se of at least three independent experiments. Columns with different superscripts differ significantly (P < 0.05).

Fig. 7.

Expression of different members of the MKP family upon LH stimulation. Ovarian follicles were exposed to LH for 0, 1, 2, 3, and 4 h. At the end of incubation, RNA was extracted, and cDNA was synthesized and subjected to Q-PCR for MKP-1 (DUSP1), MKP-2 (DUSP4), MKP-3 (DUSP6), MKP-X (DUSP7), and PAC1 (DUSP2) transcripts. The graph shows means ± se of three independent experiments (*, P < 0.03; **, P < 0.005).