Luteinizing hormone-dependent activation of the epidermal growth factor network is essential for ovulation

Abstract

In the preovulatory ovarian follicle, mammalian oocytes are maintained in prophase meiotic arrest until the luteinizing hormone (LH) surge induces reentry into the first meiotic division. Dramatic changes in the somatic cells surrounding the oocytes and in the follicular wall are also induced by LH and are necessary for ovulation. Here, we provide genetic evidence that LH-dependent transactivation of the epidermal growth factor receptor (EGFR) is indispensable for oocyte reentry into the meiotic cell cycle, for the synthesis of the extracellular matrix surrounding the oocyte that causes cumulus expansion, and for follicle rupture in vivo. Mice deficient in either amphiregulin or epiregulin, two EGFR ligands, display delayed or reduced oocyte maturation and cumulus expansion. In compound-mutant mice in which loss of one EGFR ligand is associated with decreased signaling from a hypomorphic allele of the EGFR, LH no longer signals oocyte meiotic resumption. Moreover, induction of genes involved in cumulus expansion and follicle rupture is compromised in these mice, resulting in impaired ovulation. Thus, these studies demonstrate that LH induction of epidermal growth factor-like growth factors and EGFR transactivation are essential for the regulation of a critical physiological process such as ovulation and provide new strategies for manipulation of fertility.

FIG. 1.

Delayed onset of oocyte meiotic maturation and reduced cumulus expansion in Areg^−/− mice. (A) Time course of breakdown of the oocyte nuclear membrane (GVBD) in wild-type and Areg^−/− mice. In vivo-stimulated ovaries were collected at the indicated times after hCG, and large antral follicles were punctured to release the oocytes. Data are shown as means ± SEM, and three to seven females were used at each time point. The percentage of oocytes that resumed meiosis was significantly lower in Areg^−/− mice at 3 h and 4 h after hCG than in WT/Areg^+/− females. (B) Pattern of gene expression in Areg^−/− COCs. Semiquantitative RT-PCR analysis was performed to evaluate Ptgs2, Tnfaip6, and Has2 mRNA expression levels in COCs isolated from Areg^+/− and Areg^−/− mice stimulated with hCG for 3 h. Data are shown as the means ± SEM of the relative expression levels for Ptgs2, Tnfaip6, or Has2 normalized to Rpl19 (Areg^+/−, n = 7; Areg^−/−, n = 10). (C) Morphological analysis of cumulus expansion in Areg^−/− mice. The degree of cumulus expansion was evaluated for COCs in preovulatory follicles of ovaries stimulated with hCG for 9 h by use of a subjective 0 to +4 scoring system (55). Ovaries from four different females were analyzed for each genotype. The data are shown as means ± SEM. *, P < 0.001. (D) Histology of COCs in Areg^+/− and Areg^−/− preovulatory follicles 9 h after hCG and Ereg^−/− follicles 8 h after hCG. In these selected COCs, the metaphase spindle is visible in the oocytes. Areg^+/− COCs are well expanded, whereas COCs from Areg^−/− and Ereg^−/− mice appear moderately or poorly expanded.

FIG. 2.

Development of ovarian follicles up to the preovulatory stage in Areg^−/− Egfr^wa2/wa2 mice. (A) Histological analyses of PMSG-primed Areg^−/− Egfr^wa2/wa2 mouse ovaries showed that many large antral follicles develop in response to the hormone stimulation. (B) Spontaneous maturation of oocytes (scored as percent GVBD) isolated from preovulatory follicles of PMSG-primed Areg^−/− Egfr^wa2/wa2 mice occurs in culture in vitro at a rate similar to that seen with wild-type oocytes (n = 4 for each genotype). (C) Similar levels of Lhcgr mRNA are expressed in Areg^−/− Egfr^wa2/wa2 and wild-type ovaries as determined by semiquantitative RT-PCR. Data are means ± SEM of the relative expression levels for Lhcgr normalized to Rpl19 (n = 3 for each genotype). (D) Preovulatory follicles from Areg^−/− Egfr^wa2/wa2 mice cultured in the presence of rLH for 30 min respond with a normal increase in cAMP. Data represent the means ± SEM of the results of three separate experiments.

FIG. 3.

Impaired EGFR activation and oocyte meiotic resumption in Areg^−/− Egfr^wa2/wa2 mice. (A) LH-induced EGFR phosphorylation in Areg^+/+ Egfr^+/+, Areg^+/+ Egfr^wa2/wa2, and Areg^−/− Egfr^wa2/wa2 preovulatory follicles. Proteins extracted from follicles cultured in the absence or presence of 5 IU rLH for 2 h were immunoprecipitated using an anti-EGFR antibody, and Western blotting was performed using an EGFR-specific phosphoantibody. To control for protein loading, the same membrane was reprobed using an anti-EGFR antibody. Data represent the means ± SEM of the results of three separate experiments. P < 0.05 compared to wild-type (a) and Areg^+/+ Egfr^wa2/wa2 (b) mouse results. A representative blot is shown. (B) Resumption of oocyte maturation (scored as GVBD) after 4 h hCG stimulation in vivo was evaluated for immature mice of the indicated genotypes. Results represent the means ± SEM of the results for each group. The number of mice per group is indicated above each bar. The letters a and b correspond to P < 0.05 and P < 0.001, respectively, compared to wild-type results, and c indicates P < 0.05 compared to Areg^−/− results.

FIG. 4.

Defective cumulus expansion in Areg^−/− Egfr^wa2/wa2 mice. (A) Semiquantitative RT-PCR results show significantly decreased expression levels for Ptgs2, Tnfaip6, and Has2 in COCs from Areg^−/− Egfr^wa2/wa2 mouse ovaries 3 h after hCG compared to wild-type control results. Data represent means ± SEM of the expression levels for Ptgs2, Tnfaip6, or Has2 normalized to Rpl19 (n = 3 for each genotype). (B) In situ hybridization analysis shows high expression and localization of Ptgs2 mRNA in mural granulosa cells (GC) and cumulus cells (CC) of preovulatory follicles from wild-type ovaries 3 h after hCG (a and b). The signal is decreased in GCs and is below the threshold of detection in CCs of Areg^−/− Egfr^wa2/wa2 follicles (c and d) (Oo, oocyte). (C) The degree of cumulus expansion for COCs in Areg^−/− Egfr^wa2/wa2 mouse ovaries 9 h after hCG was evaluated as described in Materials and Methods. A large proportion of COCs were poorly expanded or unexpanded. Data represent the mean percentage of COCs scored ± SEM (n = 4 females; *, P < 0.001). The degree of cumulus expansion in Areg^+/− mice (see Fig. 1C) is included for comparison. (D) Histology of COCs with GV and GVBD oocytes (left and right panels, respectively) from Areg^−/− Egfr^wa2/wa2 mouse ovaries 9 h after hCG.

FIG. 5.

Impaired ovulation and luteinization in Areg^−/− Egfr^wa2/wa2 mice. (A) Superovulation of wild-type and mutant mice. Ereg^−/−, Areg^+/+ Egfr^wa2/wa2, and Areg^−/− Egfr^wa2/wa2 mice ovulated significantly fewer oocytes than wild-type and Areg^−/− females. The number of females used per group is indicated in parentheses above the bars. Data represent means ± SEM; * indicates P < 0.001 compared to either wild-type (Areg^+/+ Egfr^+/+) or Areg^−/− values. (B) Images of ovulated COCs produced using Hoffman interference microscopy. Panels a and b show ovulated, well-expanded complexes from a representative wild-type mouse. In contrast, both expanded and poorly expanded complexes were observed in the few COCs that were ovulated by Areg^+/+ Egfr^wa2/wa2 mice (c; at higher magnification in panel d). Structures resembling ovulated COCs from Areg^−/− Egfr^wa2/wa2 mice (e and f) appeared poorly expanded. (C) Histology of Areg^−/− Egfr^wa2/wa2 ovaries 48 h after hCG stimulation. Many antral follicles contain entrapped, unexpanded COCs (panels a and c) with oocytes still in the GV stage (shown at higher magnification in panels b and d). Somatic cells from antral follicles appeared either nonluteinized (a and b) or partially luteinized (c and d), with morphologically nonluteinized granulosa cells (black arrows) lining the antrum and luteal cells (white arrows) in the outer cell layers. The asterisks mark entrapped GV oocytes. (D) In situ hybridization analysis localizes Cyp11a1 mRNA in the corpora lutea of a wild-type ovary (a and b) and in luteinized cells of antral follicles from an Areg^−/− Egfr^wa2/wa2 ovary (c and d) 48 h after hCG stimulation. Signal for Cyp11a1 is absent from non-terminally differentiated somatic cells.