A Murine 5-Fluorouracil-Based Submyeloablation Model for the Study of Bone Marrow-Derived Cell Trafficking in Reproduction

Abstract

Bone marrow (BM)-derived cells (BMDCs) contribute to endometrial regeneration. Our objective was to develop a nongonadotoxic mouse BM transplant (BMT) model using 5-fluorouracil (5-FU) for investigating BMDCs trafficking in reproduction. Female C57BL/6J mice received either single (CTX-1) or paired (CTX-2) 5-FU (150 mg/kg) dose, or single (CTX-1+SCF) or paired-dose (CTX-3+SCF) 5-FU with stem cell factor (SCF). Control mice received BMT only or saline. BM cells (20 × 10⁶) from transgenic green-fluorescent protein (GFP) mice were injected iv. For fertility experiment, mice were mated on day 28 after BMT. Alternatively, mice were killed 1 month after BMT and BMDCs recruitment to the uterus was determined. Mice receiving 5-FU ± SCF showed intact ovarian function and fertility. CTX-3+SCF resulted in greatest BM donor chimerism at 1 month (∼45%). Flow cytometry analysis demonstrated that 6.6% of total uterine cells in CTX-3+SCF mice were GFP+ BMDCs. Remarkably, this was about 40- and 80-fold greater than BMDCs in uterus of CTX-1 or BMT only mice (6.6% vs 0.16% vs 0.08%, respectively, P < .001). Immunohistochemical analysis showed that BMDCs in the uterus were mostly localized to the endometrial stroma (71.8%). The majority of endometrial BMDCs colocalized with the pan-leuokocyte CD45 marker (58.5%), but 41.5% were CD45-negative. Cytokeratin and CD31 staining showed that the GFP+CD45- cells were not epithelial or endothelial, confirming their stromal identity. We demonstrate that paired-dose 5-FU regimen results in efficient BM donor chimerism while maintaining ovarian function and fertility. This model could be used for studying BMDCs trafficking to the uterus in various reproductive physiological and pathological conditions.

Figure 1

Toxicity of various 5-FU-based submyeloablation regimens. A, A schematic depicting the different regimens used for BM conditioning. Mice received ip injection of either PBS or 5-FU on days −6 and −1 before BMT. In addition, SCF was administered by 3 separate 50-μg/kg ip injections at 21 and 9 hours before and 3 hours after 5-FU dose. B, Percentage change in weight over time in the different BM conditioning groups demonstrating a significant decrease in weight in the CTX-2 group compared with CTX-1, BMT, and PBS control groups. C, Percentage change in weight over time in the CTX-3+SCF, CTX-1+SCF, and PBS control groups. D, Kaplan-Meier survival curve showing decreased survival in CTX-2 mice compared with all other groups; n = 29 for CTX-2, n = 14 for all other groups. Data are presented as mean ± SEM; *, P < .01 and **, P = .005 for CTX-2 vs all other groups.

Figure 2

Flow cytometry analysis of donor chimerism. A, Representative images of flow cytometry analysis of peripheral blood cells on day 28 after BMT demonstrating the percentage of GFP-positive cells in the circulation in the various BM conditioning regimens. B, Mean %GFP-positive circulating cells on days 7, 14, 21, and 28 after BMT; n = 6 mice assayed in each group. Data in graph are presented as mean ± SEM; *, P < .05 for CT-3+SCF vs all other groups; +, P < .01 for CTX-1+SCF and CTX-3+SCF groups vs CTX-1 and CTX-2, respectively (P < .05).

Figure 3

Effects of various 5-FU-based submyeloablation regimens on mice fertility. Mice were mated with proven-fertility males after a 21-day recovery after BMT. A, Pregnancy rate. B, Mean number of implantation sites. C, Mean number of viable fetuses. D, Mean pregnancy resorption rate per mouse; n = 12–14 mice per group. Data are presented as mean ± SEM or as percentages, as appropriate. *, P < .05 vs all other groups.

Figure 4

BMDCs recruitment to the uterus at 1 month after BMT. A and B, Representative images (A) and graph (B) of flow cytometry of total uterine cells showing the percentage of GFP-positive BMDCs found in the uterus in CTX-3+SCF, CTX-1, BMT, and PBS groups; n = 6–8 mice per group. Data are presented as mean percentage ± SEM; *, P = .001 vs all other groups. C, Immunostaining of uterine sections using anti-GFP antibody (brown) showing abundance of GFP-positive BMDCs in uterus of CTX-3-SCF-treated mice. Insets are higher magnification of the dashed rectangles. Scale bar, 100 μm.

Figure 5

BMDCs give rise to stromal cells but not endothelial or epithelial cells in the uterus at 1 month after BMT. A–D, Fluorescence confocal microscopy analysis of uterine tissue sections. Uterine tissues of PBS control (A) or CTX-3+SCF mice (B–D) were stained with anti-GFP antibody (green) and costained with either anti-CD45 (pan-leukocyte marker) antibody (red) (A and B), CD31 (endothelial marker) antibody (red) (C), or cytokeratin (epithelial marker) antibody (red) (D). Nuclei were stained by DAPI and are shown in blue. Insets are higher magnification of areas within the section. White arrows point to BM-derived leukocytes (GFP-positive/CD45-positive). White arrowheads point to nonleukocyte BMDCs (GFP-positive/CD45-negative), which also did not colocalize with CD31 (C) or cytokeratin (D) markers, indicating their stromal identity. Scale bar, 50 μm.