Characterization of the ovarian and reproductive abnormalities in prepubertal and adult estrogen non-responsive estrogen receptor alpha knock-in (ENERKI) mice

Abstract

Estrogen non-responsive estrogen receptor alpha (ERalpha) knock-in (ENERKI) mice have a mutation (glycine 525 to leucine, G525L) in the ligand-binding domain of ERalpha. The mutant ERalpha protein has a significantly lower affinity and response to endogenous estrogens, while not altering growth factor activated ligand-independent pathways. ENERKI females demonstrated signs of early follicle development as determined by a significant increase in antral follicle formation by 20 days of age. Adult ENERKI females were infertile, had hemorrhagic ovarian follicular cysts, and failed to develop corpora lutea in response to a superovulation regimen. These results illustrate the importance of ERalpha ligand-induced signaling for ovarian development and for estrogen feedback on the hypothalamus and pituitary. Although ERalpha ligand-induced signaling by endogenous estrogens is lost in ENERKI females, the ERalpha selective agonist propyl pyrazole triol (PPT), a synthetic nonsteroidal compound, is still able to activate G525L ERalphain vivo to increase uterine weight. To test whether PPT could restore ligand-dependent receptor activation, ENERKI females were treated with PPT and evaluated for spontaneous ovulation, ovarian hemorrhagic cysts, and LH serum levels. Daily PPT treatments beginning on day 4 of life prevented formation of ovarian hemorrhagic cysts in adult ENERKI animals. In accordance with this result, preputial gland weight and LH levels were also lowered in these animals, indicating PPT treatments most likely led to restoration of ERalpha negative feedback of the hypothalamic-pituitary axis.

Figure 1

Ovarian follicle development in prepubertal ENERKI mice. Ovarian tissue H&E staining from immature 18-20 day old wild type (A), heterozygous (B), and ENERKI (C) mice (100X). The number of secondary follicles, antral follicles, and early cysts were counted in both ovaries in three representative sections per animal. (D) Average number of follicles per section examined for each genotype. ENERKI females had a higher number of antral and cyst-like follicles than their wild type (WT) and heterozygous (HET) littermates (D). a, ENERKI value significantly different from WT value (p<0.01). b, ENERKI value significantly different from WT and HET value (p<0.01).

Figure 2. Superovulation Experiments

ENERKI ovaries fail to develop corpora lutea after superovulation. 12-week-old animals were injected with PMSG and hCG in a standard superovulation regime. Ovarian tissue H&E staining from superovulated 12 week old animals indicated wild type (A) ovaries contained many corpora lutea (CL) and healthy follicles, while ENERKI (B) ovaries had no corpora lutea and the majority contained large hemorrhagic cysts (100X). Immature animals were also superovulated. Wild-type animals generated corpora lutea (C), while immature ENERKI mice (D) has partially luteinized, hemorrhagic structures that produced oocytes in the oviduct but did not fully form a corpora lutea.

Figure 3. Long-Term PPT Injections

Long-term daily PPT injections prevent formation of ovarian hemorrhagic cysts in ENERKI mice. Groups of 4-5 ENERKI females were injected subcutaneously with vehicle from 4 days of age (A), 10,000 μg/kg PPT from 4 days of age (B), or 10,000 μg/kg PPT from 3.5 weeks of age (C), everyday until 8 weeks of age. A subset of animals injected with 10,000 μg/kg PPT from 3.5 weeks of age was injected with 100,000 μg/kg PPT every fourth day to mimic the estrous cycle surge (D). Ovarian tissue H&E staining (100X) revealed daily vehicle (A) and 10,000 μg/kg PPT treatments from 3.5 weeks of age (C) exhibited the same phenotype. Hemorrhagic cyst formation was prevented in ENERKI mice treated with 10,000 μg/kg PPT from 4 days of age (B) or 100,000 μg/kg PPT surges every fourth day (D).