Uterine cysts in female mice deficient for caveolin-1 and insulin-like 3 receptor RXFP2

Abstract

Gene mutations of insulin-like 3 (INSL3) peptide or its G protein-coupled receptor RXFP2 (relaxin family peptide receptor 2) lead to cryptorchidism. The role of INSL3 in adult females is less known, although INSL3 expression has been described in female reproductive organs. Caveolin-1 (CAV1), the main component of caveoli cell membrane invaginations, has been shown to play an important role in epithelial organization and stromal-epithelial interactions. We created a null allele of Cav1 mice by deleting its second exon through embryonic stem cell targeting. Immunohistochemical analysis demonstrated that CAV1 expression was primarily localized to endothelial blood vessel cells and the myometrium uterus, whereas the strongest expression of Rxfp2 was detected in the endometrial epithelium. By 12 months of age approximately 18% of Cav1-/- females developed single or multiple dilated endometrial cysts lined by a flattened, simple low epithelium. A deficiency for Rxfp2 on Cav1-deficient background led to more than a 2-fold increase in the incidence of uterine cysts (54-58%). Appearance of cysts led to a severe disorganization of uterine morphology. We have found that the cysts had an increased expression of β-catenin and estrogen receptor β in endometrial stromal and epithelial cells and increased epithelial proliferation. An analysis of simple dilated cysts in human patients for CAV1 expression did not show appreciable differences with control regardless of menstrual phase, suggesting an involvement of additional factors in human disease. The results of this study suggest a novel synergistic role of INSL3/RXFP2 and CAV1 in structural maintenance of the uterus.

Fig. 1.

Generation of mice with a mutant allele of the Cav1 gene. A targeting construct was prepared by insertion of loxP in NruI and floxed Neo cassette in NdeI restriction sites on both sides of exon 2. Neo, Neomycin resistance cassette; TK, herpes virus thymidine kinase minigene; the loxP sites are shown as the thick arrows; black boxes represent Cav1 exons. The wild-type chromosome is shown on top. After homologous integration the targeting construct is integrated into genomic DNA. After Cre-mediated recombination in ES cells, the knockout allele was produced (bottom). To verify the deletion of the second exon in ES clones, we used genomic PCR with primers located on both sides of the floxed region. The mutant allele (Cav1-) was detected through successful amplification of DNA fragment (lane 2), whereas there was no amplification in wild-type genomic DNA (lane 1). M, 1 Kb marker.

Fig. 2.

The expression of Cav1 and Rxfp2 in mouse uterus. A, Immunohistochemical localization of CAV1 in wild-type uterus and an absence of staining in Cav1−/− uterus. Females (1 yr old) were used. Strong signal was detected in endothelial cells of the blood vessels and myometrium. Note an absence of staining in glandular endometrial epithelium. B, The β-galactosidase staining showing the Rxfp2-promoter-dependent expression of iCre recombinase in Rxfp2-iCre, R26R transgenic mice (left). No staining was detected in uterus of R26R controls (right). The most prominent blue staining is located in endometrial glandular epithelial cells and luminal epithelium. Two month old females were used. Magnification bar, 100 μm.

Fig. 3.

Endometrial cyst development in Cav1-deficient mice. A, Polycystic uterus from 12-month-old Cav1−/−, Rxfp2−/+ female (right image). Multiple cysts filled with blood are visible in both uterine horns. Normal uterus of wild-type female is shown on the left. Magnification bar, 10 mm. B, Cross-sections of representative uterine abnormalities in Cav1−/−. Top left image is a wild-type uterus (no. 142). Endometrial cysts in Cav1−/− (nos. 8 and 58), Cav1−/−, crsp/+ (nos. 73 and 102), Cav1−/−, Rxfp2+/− (nos. 60, 145, and 147). All animals are 11–13 months old. Note the presence of eiosinophilic secretion or blood cells in some cysts. Magnification bar, 500 μm. C, Expression of β-catenin (boxed areas are shown at higher magnification in a second row), Ki67, ESR1, and ESR2 in 11- to 13-month-old wild-type, Cav1−/−, and Cav1−/−, Rxfp2−/+ uteri. Note a significant increase of β-catenin, Ki67, and ESR2-positive cells. Magnification bars, 100 μm, row 1; 20 μm, rows 2–5.

Fig. 4.

An increase of epithelial endometrial cell proliferation in cystic uteri. The proliferation index was calculated as a percentage of Ki67-positive cells in normal (NE) and cystic (CE) epithelium in uteri without cysts (NC) or with cysts (CU). Genotype of the animals is indicated. WT, Age-matched wild-type females. Each group represented by three to six 11- to 13-month-old animals. Differences between NE in animals without cysts and NE or CE in animals with cysts are significant (P < 0.01).

Fig. 5.

CAV1 expression in normal human endometrium and in endometrium with dilated cysts. There is a strong CAV1 staining in endothelial cells of blood vessels, weak CAV1 staining in stromal endometrial cells, and no expression in the endometrial glandular epithelium. Normal endometrium is on the left, endometrium with dilated cysts is on the right. Magnification bars, 200 μm, top; 50 μm, bottom.