Changes in the mouse estrus cycle in response to BRCA1 inactivation suggest a potential link between risk factors for familial and sporadic ovarian cancer

Abstract

Menstrual cycle activity is the most important risk factor for sporadic serous ovarian carcinoma, whereas a germ-line mutation in BRCA1 is the most important risk factor for the familial form. Given the rarity of BRCA1 mutations in sporadic ovarian cancers, we hypothesized that BRCA1 influences the menstrual cycle in a way that mimics the factors underlying sporadic ovarian cancer predisposition, making BRCA1 mutations redundant in such cancers. We compared the length of each phase of the estrus cycle (equivalent to the human menstrual cycle) and of circulating levels of estradiol in control mice and in mice carrying a Brca1 mutation in their ovarian granulosa cells, two thirds of which develop ovarian or uterine epithelial tumors. We also compared the length of the different phases of the cycle in mutants that subsequently developed tumors with those in mutants that remained tumor-free. Mutant mice as well as oophorectomized wild-type mice harboring mutant ovarian grafts showed a relative increase in the average length of the proestrus phase of the estrus cycle, which corresponds to the estrogen-dominated follicular phase of the human menstrual cycle. Total circulating levels of estradiol were also increased in mutant mice injected with pregnant mare serum gonadotropins. The relative increase in proestrus length was highest in mutant mice that subsequently developed reproductive epithelial tumors. We conclude that loss of a functional Brca1 increases murine ovarian epithelial tumor predisposition by increasing estrogen stimulation in the absence of progesterone, recapitulating conditions associated with sporadic ovarian cancer predisposition in humans.

Figures

Figure 1. Changes in vaginal cytology associated with the different stages of the estrus cycle

Daily vaginal washings obtained as explained in the methodology section from a single mouse were stained with Papanicolou (PAP) stain. The illustration shows photographs representative of each stage of the estrus cycle. Diestrus is characterized primarily by inflammatory cells with few, if any, immature epithelial cells. Proestrus shows turquoise epithelial cells of intermediate maturity, which become more mature orange cells toward the end of this phase. Estrus is characterized by fully mature orange cells lacking a nucleus. Metestrus is characterized by intermediate and fully mature cells admixed with inflammatory leukocytes.

Figure 2. Activity of the Fshr promoter in a subset of anterior pituitary cells

A: DNA extracted from ovary, pituitary gland, or spinal cord was enzymatically amplified using primers that can distinguish between rearranged and intact forms of the floxed Brca1 allele (11). The mice carried either one (+/−) or two (−/−) mutant Brca1 alleles. A 684 bp fragment corresponding to the mutant Brca1 allele was amplifiable from ovarian and pituitary tissues, but could not be amplified from spinal cord. A 530 bp fragment representing the intact (unrearranged) floxed Brca1 allele was amplified from all tissues. A 470 bp fragment representing the wild type gene was only seen in mice carrying a single floxed Brca1 allele. B: The upper photograph shows the pituitary of a Fshr-Cre;Brca1 flox/flox;R26R mouse stained for LacZ. The dark color indicates Beta-galactosidase activity, which in turn is indicative of Fshr promoter activity in the Fshr-Cre transgene. Focal promoter activity is also appreciated in the photomicrograph of a similar pituitary (bottom). Bar: 100 microns.

Figure 3. Demonstration of the functionality of transplanted ovaries

The illustration at the top shows a whole mount photomicrograph of a kidney with an attached subcapsular ovarian transplant. The area within the rectangle is magnified in the bottom left panel, showing a secondary ovarian follicle (arrow) that is further magnified in the photograph shown on the right. The presence of such follicles further attests to the functionality of transplanted ovaries.