Female mice expressing constitutively active mutants of FSH receptor present with a phenotype of premature follicle depletion and estrogen excess

Abstract

Strong gain-of-function mutations have not been identified in humans in the FSH receptor (FSHR), whereas such mutations are common among many other G protein-coupled receptors. In order to predict consequences of such mutations on humans, we first identified constitutively activated mutants of the mouse (m) Fshr and then expressed them under the human anti-Müllerian hormone promoter in transgenic mice or created knock-in mutation into the mouse genome. We show here that mutations of Asp580 in the mFSHR significantly increase the basal receptor activity. D580H and D580Y mutations of mFSHR bind FSH, but the activity of the former is neither ligand-dependent nor promiscuous towards LH/human choriogonadotropin stimulation. Transgenic expression of mFshr(D580H) in granulosa cells leads to abnormal ovarian structure and function in the form of hemorrhagic cysts, accelerated loss of small follicles, augmented granulosa cell proliferation, increased estradiol biosynthesis, and occasional luteinized unruptured follicles or teratomas. The most affected mFshr(D580H) females are infertile with disturbed estrous cycle and decreased gonadotropin and increased prolactin levels. Increased estradiol and prolactin apparently underlie the enhanced development of the mammary glands, adenomatous pituitary growth, and lipofuscin accumulation in the adrenal gland. The influence of the mFSHR(D580Y) mutation is milder, mainly causing hemorrhagic cysts in transgenic mFSHR(D580Y) and mFSHR(D580Y) -knock-in mice. The results demonstrate that gain-of-function mutations of the FSHR in mice bring about distinct and clear changes in ovarian function, informative in the search of similar mutations in humans.

Figure 1

A, Signaling activity of the mFSHR variants in vitro as measured by their cAMP responses, i.e. cre-luciferase reporter gene activation, and adjusted for their ligand binding capacity without hormonal stimulation. B, cAMP response of the mFSHR variants after stimulation with FSH. C, Activation of the mFSHR variants by LH and hCG. Open bars in all panels depict reporter gene activation without ligand stimulation. Filled bars in panel B depict (from left to right) the response to 1, 3, 10, 30, and 100 IU/liter FSH stimulation. Light and dark gray bars in panel C depict (left to right) stimulations with 0.05, 5, and 50 IU/ml of hCG and LH, respectively. The pSG-5 mock-transfected samples were stimulated with the highest concentrations of hCG and LH. RLU, relative luciferase units (luciferase activity/renilla luciferase activity). Bars, average + sd of triplicates. The data represent typical results from at least three independent experiments.

Figure 2

Gross histology of ovaries in WT (A–C) and mFshr^D580H (E–G) females at the age of 28 d (A and E), 15 wk (B and F), and 12 months (C and G) and PAS staining of an ovary of 6-month-old WT (D) and tg (H) mice. Hemorrhagic cysts are common in the ovaries of the tg mice (E–G, arrows) showing leakage of blood cells from the thecal layer into the antrum (G, inset i). Multiple simultaneously maturing antral follicles increase the size of the tg ovaries (F). In most of the old mice, large follicles are maturing, but stroma is occupied by interstitial hyperplasia (G, inset ii, and H). Minor staining with PAS is seen in the ovaries of WT mice, in which mainly zonae pellucidae and oocyte remnants present with the stain (D, inset), but in the ovaries of mFshr^D580H mice, the stroma is broadly stained with PAS (H, inset ii). The oocyte in the unruptured luteinized follicle is also encircled by PAS-positive zona pellucida (H, inset i), and the surface epithelium has thickened adjacent to the PAS-stained areas (H, inset ii, arrow). AF, antral follicle; CL, corpus luteum. Scale bars, Main frames, 200 μm; D, inset, and H, inset ii, 100 μm; G, inset, 50 μm; H, inset i, 20 μm. D and H, Blue staining, nuclei; purple staining, PAS-positive glycogen.

Figure 3

Classification and numbers of ovarian follicles of 2-month-old (A) and 6-month-old (B) WT and mFshr^D580H mice reveal accelerated loss of small follicles at age of 6 months in the latter. The bars represent average + sem of four independent samples. Open bars, WT; filled bars, tg mice; primrd & trans, primordial and transition; prim & prim+, primary and primary plus; *, P < 0.05; ***, P < 0.005.

Figure 4

Macroscopic (A and B) and microscopic (C–E) presentation of ovarian teratomas in mFshr^D580H females. The right ovary is hemorrhagic (A, arrowhead), characteristic of mFshr^D580H females, whereas the left one (A, arrow, and B) presents an advanced teratoma. Squamous and cuboidal lined cysts (C) and osteogenesis (D) are typically seen in the teratomas. Area of poorly differentiated and necrotic immature teratoma (E) contains mitotic cells (inset, arrows). Scale bars, Main frames C–E, 100 μm; inset, 100 μm.

Figure 5

E2 biosynthesis in mFshr^D580H females and their WT littermates. A, Serum E2 concentrations in WT and tg females in the age groups 2–3, 6–8, and 11–13 months. B, Quantitative RT-PCR analysis of expression of Cyp19a1; and C, its correlation to the mFshr^D580H expression in tg females. D, Quantitative RT-PCR analysis of expression of Hsd17b1 in WT and tg females. E, Serum P4 concentrations in aging WT and tg females. The samples for panel C have been collected from randomly cycling mice, and for the other panels from metestrus or diestrus. Relative gene expressions have been calculated as target gene mRNA copy number/mRlp7 mRNA copy number. Numbers of samples analyzed has been shown below each column. Bars, average + sem. In all panels: open bar, WT; filled bar, tg; *, P < 0.05.

Figure 6

Histology and function of the pituitary and mammary glands in mFshr^D580H female mice. The pituitary of tg females is enlarged (A–C), contains blood-filled cavities (A, inset i) and mitotic cells (arrows in A, inset ii). PRL production is increased in tg females (D). Mammary gland of a 6-month-old WT mouse demonstrates basal branching (E), whereas the tissue of a tg virgin mouse shows intense lobulo-alveolar growth (F and inset). Serum FSH (G) and LH (H) concentrations are decreased in mFshr^D580H but not in mFshr^D580Y females during metestrus-diestrus. The samples with level under detection limit (0.1 ng/ml for FSH and 40 pg/ml for LH) have been calculated with that value. C, D, G, and H, Bars, average ± sem and numbers of samples analyzed are shown below graphs. G and H, Open bar, WT; filled bar; mFshr^D580H mice; gray bar, mFshr^D580Y mice. ln, lymphatic node. Scale bars, Main pictures A, B, E, and F, 500 μm; A, inset i, 100 μm; A, inset ii, 20 μm; F, inset, 100 μm. **, P < 0.01; ***, P < 0.005.