Granulosa Cell-Specific Inactivation of β-Catenin Alters Aromatase Production and Normal Folliculogenesis

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We recently reported that FSH signaling to induce Cyp19a1 (aromatase), the rate-limiting enzyme in estrogen biosynthesis, requires the co-activator β-catenin (Ctnnb1) in a primary rat granulosa cell model. The purpose of the following investigation was to ascertain the physiological significance of β-catenin to granulosa cell maturation and folliculogenesis in an in vivo mouse model. To achieve this purpose, Ctnnb1flox/flox mice were intercrossed with mice that express Cre recombinase under the control of Amhr2 (Ctnnb1flox/flox;Amhr2Cre/+). Amhr2 is expressed along the Mûllerian ducts and in developing ovaries at embryonic day 13.5. Postnatally granulosa cells express Amhr2 specifically in developing follicles. Conditional deletion of the β-catenin gene in 8 week-old females resulted in lower uterine weights when compared to their wild-type counterparts, consistent with the recognized requirement for β-catenin in reproductive tract development. However, conditional deletion of the β-catenin gene did not affect the time to puberty (d 33). No obvious ovarian phenotype was detected between conditional knockouts and wild-type mice at 8 weeks of age. Follicles appeared to undergo maturation to the preovulatory phenotype and to form normal numbers of corpora lutea compared to wild-type mice. Ctnnb1flox/flox;Amhr2Cre/+ mice were found to be infertile after they failed to produce a litter within six months when paired with proven male studs, a result most likely attributable to aberrant reproductive tract development. Unexpectedly, pituitary Fshb mRNA but not Lhb mRNA levels were greater in conditional knockouts compared to wild-type mice. Similarly, the FSH-regulated ovarian genes Inhα and Lhr were also up-regulated in conditional knockout mice compared to wild-type mice. Serum estradiol concentrations and Cyp19a1 mRNA expression also tended to be elevated in knockout compared to wildtype mice. However, total ovarian β-catenin mRNA levels were not reduced in conditional knockout mice. To focus on the effect of β-catenin in granulosa cells we therefore used an in vitro knockout model for β-catenin in primary cultures of mouse granulosa cells. Granulosa cells from Ctnnb1flox/flox were isolated and exposed to a Cre recombinase adenovirus, resulting in a marked reduction of β-catenin mRNA and protein. Deletion of β-catenin in granulosa cells resulted in loss of the ability of FSH to induce Cyp19a. Consequently, estradiol concentrations in cell culture media were higher in FSH-stimulated control granulosa cells compared to FSH-stimulated granulosa cells in which β-catenin was deleted (15.4 ± 3.2 pg/ml and 7.6 ± 2.3 pg/ml, respectively). Collectively, the data herein indicate that β-catenin plays a role in folliculogenesis in the developing ovary, specifically enabling granulosa cells to respond to FSH by expressing Cyp19a1. In addition, our results indicate that β-catenin, normally viewed as an essential component of the canonical WNT signaling pathway, is also required for mediating hormonal signaling through G-protein coupled receptors that regulate the production of estrogen. Supported by a minority fellowship to JAHG (P01 HD21921 to MHD).