PMC:5608921 / 6947-10069 JSONTXT

{"project":"2_test","denotations":[{"id":"28846079-12525422-79081914","span":{"begin":2357,"end":2359},"obj":"12525422"}],"text":"Increased number of cycles and longer proestrus–estrus phase in Tg(K6b-E6/E7) mice\nIn order to obtain indications of estrous cycle progression, we compared Tg(K6b-E6/E7) and WT mice regarding the number of cycles, as determined by the frequency of sequential proestrus–estrus phases, and the time staying at the growth phases, as determined by the frequency of proestrus and estrus, both within a defined number of days (19 days). Notably, the frequency of cycles (Figure 3a) and, apparently, the incidence of growth phases (Figures 3b–d) were higher in Tg(K6b-E6/E7) than in WT mice. Nonetheless, the initiation of each cycle was still dependent on estradiol as cycling was blocked in both Tg(K6b-E6/E7) and WT mice treated with raloxifene (Figures 3c and d).\nCytological analysis after a short-term β-estradiol (E2) treatment showed a tendency of mice to stay in the growth phases of the estrous cycle (Figures 3c and d). This result suggests a possible effect of estradiol on epithelium during the growth phases. To test this hypothesis, mice were treated with raloxifene once proestrus had initiated and, then, cell proliferation evaluated by the presence of Ki67 (Figure 3e). Despite the higher number of suprabasal proliferating cells due to oncogene expression, reduction in the number Ki67+ cells was observed in both WT and Tg(K6b-E6/E7) mice with a similar effect in basal/parabasal and suprabasal cells (Figure 3f); no indication of cell death was detected in the presence of raloxifene (Supplementary Figure S1). These results confirm the critical role of estradiol in epithelial growth and support the cooperation with oncogenes during the proestrus–estrus phase.\nIn contrast with short-term E2 treatments, proliferating cells in suprabasal layers of WT cervical epithelia were detected (about 28% of total Ki67+ cells) after long-term treatments (\u003e1.5 months) and, as expected, many more of those were detected (about 44% of total Ki67+ cells) in the cervical epithelia of Tg(K6b-E6/E7) mice (Figure 4a). Accordingly, long-term E2 treatments thickened the epithelia of the transformation zone of WT cervix (n=4), though a more pronounced growth in this region occurred in Tg(K6b-E6/E7) mice (n=3; Figure 4b). Interestingly, as a possible indication of a precancerous condition, koilocyte-like cells (i.e., squamous cells with a clear cytoplasm22) were found in the grown cervical epithelia of both WT and Tg(K6b-E6/E7) mice, with an evident increased number in the latter (Figure 4c). In addition, a high frequency of bi-nucleated cells was found in vaginal smears of E2-treated Tg(K6b-E6/E7) mice (Figure 4d). In WT mice, even after 9 months of E2 treatment, hyperplasia was the major phenotype of cervical epithelium. In contrast, in Tg(K6b-E6/E7) mice, hyperplasia and an irregular epithelium resembling a dysplasic tissue were frequently observed even in the absence of the hormone, as it was described above, but E2 treatment notoriously exacerbated the hyperplastic phenotype showing deep dysplasic protrusions and epithelial islands that commonly characterize the carcinoma in situ (Figures 4c and e)."}