Conditional deletion of beta-catenin mediated by Amhr2cre in mice causes female infertility

Abstract

Follicle-stimulating hormone (FSH) regulation of aromatase gene expression in vitro requires the transcriptional coactivator beta-catenin. To ascertain the physiological significance of beta-catenin in granulosa cells during folliculogenesis, mice homozygous for floxed alleles of beta-catenin were intercrossed with Amhr2cre mice. Conditional deletion of beta-catenin in 8-wk-old females occurred in derivatives of the Müllerian duct, granulosa cells and, surprisingly, in brain, pituitary, heart, liver, and tail. Female mice deficient for beta-catenin were infertile, despite reaching puberty and ovulating at the expected age, indications of apparently normal ovarian function. In contrast, their oviducts were grossly distended, with fewer but healthy oocytes. In addition, their uteri lacked implantation sites. Together, these two phenotypes could explain the complete loss of fertility. Nevertheless, although the ovary appeared normal, with serum estradiol concentrations in the normal range, there was marked animal-to-animal variation of mRNAs encoding beta-catenin and aromatase. Similarly, inhibin-alpha and luteinizing hormone receptor mRNAs varied considerably in whole ovaries, whereas pituitary Fshb mRNA was significantly reduced. Collectively, these features suggested cyclization recombination (CRE)-mediated recombination of beta-catenin may be unstable in proliferating granulosa cells, and therefore may mask the suspected steroidogenic requirement for beta-catenin. We tested this possibility by transducing primary cultures of granulosa cells from mice homozygous for floxed alleles of beta-catenin with a CRE-expressing adenovirus. Reduction of beta-catenin significantly compromised FSH stimulation of aromatase mRNA and subsequent production of estradiol. Collectively, these data suggest that FSH regulation of steroidogenesis requires beta-catenin, a role that remains hidden when tested through Amhr2cre-mediated recombination in vivo.

FIG. 1.

Genetic survey of β-catenin deletion and analysis of Cre expression. A) Reverse transcription PCR analysis was used to determine the presence of the different Ctnnb1 alleles and the Cre transgene in granulosa cells, brain, and pituitary of Ctnnb1 mutant (MUT) and wild-type (WT) mice. Gene-specific primers for the inheritance of the Cre transgene generated a 200-bp product. Primers RM41/RM42 amplify the Ctnnb1 floxed allele (324 bp) and the wild-type allele (221 bp). Amplification of a 631-bp product was used to identify the Ctnnb1 flox deleted (floxdel) allele. Results are representative of a minimum of eight animals. B) Inclusive analysis of numerous tissues depicts a strong deletion of Ctnnb1 in the brain, uterus, and oviduct (+++). Weaker deletion was detected in the heart and liver (+), and at times even in the tail DNA (+/−). Results are representative of a minimum of eight animals.

FIG. 2.

Ovaries from Ctnnb1 mutant mice have a normal histological morphology. A) Ovaries from 29- to 31-day-old wild-type and mutant mice were fixed in 4% paraformaldehyde and stained with hematoxylin and eosin. A representative cross-section of an ovary from wild-type mouse and mutant mouse (β-cat) is shown. Ovarian sections demonstrate normal development of follicles in which the majority of follicles are preantral or preovulatory. Arrow indicates a preantral follicle (original magnification ×10). B) Confocal images of wild-type and mutant mouse oocytes. The oocytes were stained with an antibody to α-tubulin to visualize the meiotic spindle (green) and DAPI to visualize the chromosomes (blue). MII-arrested wild-type (n = 226 analyzed) and β-catenin mutant (n = 63 analyzed) oocytes both illustrate formation of the first polar body (designated by the arrowhead) and the second meiotic spindle.

FIG. 3.

Infertility results from reproductive tract anomalies. A) Immature (29–31 days old) mutant mice (β-cat) exhibit only partial development of the oviduct, as evidenced by the lack of coiling typical of wild-type (WT) mice. B) Uteri were collected at 8 wk of age for wild-type and mutant mice. Uteri were trimmed of fat tissue and placed in saline until weighed. Gross morphology of a wild-type uterus exemplifies a normal, balloon-filled structure compared with the Ctnnb1 mutant uterus, which has a more flaccid appearance with the presence of fat integrated along the length of the uterus. C) Uterine wet weight in 8-wk-old wild-type and Ctnnb1 mutant females (P = 0.19; wild-type, n = 24; mutant, n = 11). D) Six days after an observed plug (plug = Day 0), 8-wk-old wild-type mice had noticeable swellings (arrow) along the uterus, indicating implantation sites (n = 5). No indication of implantation sites was observed for age-matched mutant mice (n = 2).

FIG. 4.

Variable expression of Ctnnb1 and Cyp19a1 mRNA in granulosa cells fails to change estradiol levels in Ctnnb1 mutant mice. Box-and-whisker plot distributions depict fold change of Ctnnb1 (A) and Cyp19a1 (B) mRNA expression in granulosa cells isolated from 8-wk-old mice measured by real-time PCR (wild-type, n = 6; mutant, n = 5). C) Serum estradiol concentrations measured as an indicator of Cyp19a1 activity remain unchanged between both groups (P = 0.4; wild-type, n = 11; mutant, n = 13). β-cat mut, β-catenin mutant.

FIG. 5.

Inactivation of Ctnnb1 leads to aberrant regulation of the pituitary-gonadal axis. Pituitaries and ovaries were collected from the same animals to measure gene expression changes. A single pituitary and the corresponding ovary from 8-wk-old wild-type (n = 10) and mutant (n = 7) animals were analyzed by real-time PCR for mRNAs encoding Lhb and Fshb (A) and Lhcgr and Inha (B).

FIG. 6.

CRE-mediated recombination in granulosa cells effectively reduces Ctnnb1 mRNA and protein expression. Primary granulosa cells isolated from homozygous Ctnnb1 floxed mice were transduced with EGFP or CRE adenoviruses (3 × 10¹⁰ and 5 × 10¹⁰ viral particles per milliliter [vpm], respectively). Samples were treated with FSH (100 ng/ml, 24 h) where indicated. A) Representative RT-PCR analysis determined the presence of a 324-bp floxed allele alone or in combination with the 500-bp floxed deleted (Floxed del) allele in the samples that received the CRE adenovirus. CRE was detected as the 200-bp transcript. Result is representative of four experiments. B) Reverse transcription PCR analysis also established the reduction in Ctnnb1 mRNA expression. Result is representative of four experiments. C) Western blot analysis confirmed that CRE-mediated recombination resulted in a marked reduction of Ctnnb1 protein accumulation. Blot was probed with AKT to control for protein loading. Results are representative of four experiments. VEH, vehicle; β-cat, β-catenin.

FIG. 7.

Endogenous Ctnnb1 is necessary for steroidogenesis in granulosa cells. Primary granulosa cells isolated from homozygous Ctnnb1 floxed mice were transduced with EGFP or CRE adenoviruses (3 × 10¹⁰ and 5 × 10¹⁰ viral particles per milliliter [vpm], respectively; n = 4). Samples were treated with FSH (100 ng/ml, 24 h) where indicated. CRE-mediated recombination of the Ctnnb1 gene resulted in a reduced ability of FSH to stimulate Cyp19a1, as measured by real-time PCR (upper). Estradiol production (lower) measured in the media of the cultured cells reflected the muted Cyp19a1 response. VEH, vehicle.