Our results clearly show that mice harboring a Brca1 gene knockout in their ovarian granulosa cells show a measurable increase in the average length of the proestrus phase of their estrus cycle compared to wild type mice. This was not driven by Brca1 inactivation in cells other than granulosa cells because mutant mice harboring wild type ovarian grafts showed average proestrus lengths similar to those in wild type mice.
We used homozygous Brca1 mutants in our studies in order to maximize the effects of alterations in Brca1 expression. Our results raise the possibility that human BRCA1 mutation carriers, who are heterozygous for such mutations, may experience similar alterations in their menstrual cycle due to reduction in BRCA1 gene dosage. This idea still needs to be confirmed and to this effect, studies focused on examining the consequences of heterozygous deletions of Brca1 on proestrus length and tumor predisposition are ongoing in our laboratory using our mouse model. In addition, the evidence provided in this manuscript is largely correlative and a direct demonstration that loss of a functional Brca1 increases murine ovarian epithelial tumor predisposition by increasing estrogen stimulation in the absence of progesterone is still lacking. Nevertheless, the finding that deletion of mouse Brca1, the homolog of a gene controlling familial ovarian cancer predisposition in humans, influences both tumor predisposition as well as the ovarian cycle, an important determinant ovarian cancer risk in humans, is intriguing. The human follicular phase is characterized by elevated levels of circulating estrogens relative to progesterone similarly to murine proestrus. Such hormonal exposure might be an important determinant of the site specificity of the tumors seen in human BRCA1 mutation carriers. This idea is further supported by epidemiological studies that have consistently demonstrated an association between risk of ovarian cancer and duration of postmenopausal estrogen replacement therapy (18
). In vitro
studies showing that estrogen acts as a mitogen for ovarian cancer cell lines while progesterone is growth inhibitory are also in line with this hypothesis (23
). The potential relevance of our observations to tumor predisposition in human BRCA1 mutation carriers is further underscored by our finding that mutant mice that developed tumors had higher average proestrus/metestrus + diestrus length ratios than age matched mutant littermates that remained tumor-free.
Several mechanisms could account for the changes in the dynamics of the estrus cycle in mutant animals. Estradiol biosynthesis, which is an important mediator of cycle progression, can be upregulated by reducing the expression of BRCA1 in human granulosa cells (24
). Changes in circulating estradiol levels could, by themselves, contribute to changes in the length of the pre-ovulatory phase of the cycle because levels of this hormone can influence the timing of the LH surge that triggers ovulation (25
). The finding that circulating estradiol levels were higher in mutant compared to wild type animals synchronized for their estrus cycle stage by exogenous hormonal stimulation strongly suggests that a Brca1 mutation can also influence circulating estrogen levels. It is also possible that secretion of other factors associated with the regulation of the LH surge, such as gonadotrophin surge attenuating factor (27
), which is synthesized by granulosa cells (29
), is altered in the absence of Brca1 activity. The effect of Brca1 on the level and activity of this factor as well as of hormones such as LH, inhibin, activin, and others, should be investigated.
The effects of Brca1 inactivation on the estrus cycle and on tumor predisposition seemed to be highly influenced by the age of the host animals. Not only was the magnitude of the relative increase in proestrus length higher in 7–9 month old mice than in 3–4 month old mice, but also the association between proestrus over metestrus plus diestrus ratio and tumor predisposition only reached statistical significance for ratios calculated in mice that were at least 9–10 months old. This raises the possibility that the consequences of Brca1 inactivation are magnified when the total number of ovarian follicles is decreased due to age.
Our results point to a link between the reproductive risk factors associated with the development of sporadic ovarian cancer and the genetic factors operating in human BRCA1 mutation carriers. It may be that the presence of BRCA1 mutations in sporadic ovarian carcinomas would be redundant in the presence of the strong reproductive risk factors known to be associated with such tumors, accounting for the rarity of BRCA1 mutations in non-hereditary ovarian cancers. These results also underscore the need to understand the mechanisms underlying the association between the menstrual cycle and ovarian cancer risk, which should lead to more effective prevention measures for sporadic as well as familial ovarian cancers.