Granulocyte colony-stimulating factor with or without stem cell factor extends time to premature ovarian insufficiency in female mice treated with alkylating chemotherapy

Abstract

Objective: To examine gonadal protective properties of granulocyte colony-stimulating factor (G-CSF) alone or in combination with stem cell factor (SCF) in female mice treated with high-dose alkylating chemotherapy.

Design: Experimental laboratory animal study.

Setting: Tertiary care academic hospital and research institute.

Animal(s): Six- and 8-week-old C57Bl/6 female mice.

Intervention(s): Adult female mice were treated with [1] cyclophosphamide and busulfan (CTx), [2] CTx + G-CSF/SCF, [3] CTx + G-CSF, or [4] normal saline and dimethyl sulfoxide (DMSO; vehicle control).

Main outcome measure(s): Follicle counts, microvessel density, cellular response to DNA damage, and litter production.

Result(s): G-CSF ± SCF increased microvessel density and decreased follicle loss in CTx-treated female mice compared with CTx-only treated female mice. Mice administered CTx alone exhibited premature ovarian insufficiency, with only 28% of mice producing two litters. However, 100% of mice receiving CTx with G-CSF + SCF, and 80% of mice receiving CTx + G-CSF alone produced at least three litters and 20% of mice in each group produced five litters.

Conclusion(s): Treatment of mice with G-CSF decreases chemotherapy-induced ovarian follicle loss and extends time to premature ovarian insufficiency in female mice. Further studies are needed to validate these preclinical results in humans and compare efficacy with the established GnRH analogue treatments.

Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Figures

FIGURE 1

Granulocyte colony-stimulating factor ± stem cell factor (G-CSF ± SCF) maintains primordial and early-growing follicle numbers in high-dose alkylating chemotherapy treated mice. Follicle numbers were assessed 21 days after cyclophosphamide and busulfan (CTx) treatment. Graphs represent the number of primordial (A) and early-growing (B) follicles per ovary in different treatment groups. Bars represent mean ± SEM (n = 10 mice per group; one ovary per mouse; different letters above bars indicate statistically significant differences) (P<.05). LhX8 staining indicates multiple primordial follicles in vehicle-treated controls (C), no primordial follicles in CTx-only treated ovaries (D), and few primordial follicles in CTx + G-CSF/SCF (E) and CTx + G-CSF alone (F) groups. Scale bars = 100 μm. CTx = cyclophosphamide/busulfan; CTx + G-CSF/SCF = cyclophosphamide/busulfan + granulocyte colony-stimulating factor/stem cell factor; CTx + G-CSF = cyclophosphamide/busulfan + granulocyte colony-stimulating factor.

FIGURE 2

Granulocyte colony-stimulating factor ± stem cell factor (G-CSF ± SCF) treatment decreases chemotherapy-induced gamma H2AX phosphorylation in early-growing follicles, the earliest cellular response to DNA damage. Representative panels from (A) vehicle controls, (B) cyclophosphamide and busulfan (CTx), (C) CTx + G-CSF/SCF, (D) CTx + G-CSF are shown. The percent of primordial (E) and early-growing (F) follicles exhibiting phospho-gamma H2AX staining. Bars are mean ± SEM; n = 3 mice per group; one ovary per mouse. Different letters above bars represent statistical significance (P<.05). Scale bars = 50 μm. CTx = cyclophosphamide/busulfan; CTx + G-CSF/SCF = cyclophosphamide/busulfan + granulocyte colony-stimulating factor/stem cell factor; CTx + G-CSF = cyclophosphamide/busulfan + granulocyte colony-stimulating factor.

FIGURE 3

Granulocyte colony-stimulating factor ± stem cell factor (G-CSF ± SCF) treatment increases microvessel density after chemotherapy treatment. Microvessel density was assessed 21 days after cyclophosphamide and busulfan (CTx) treatment. Examples of immunofluorescent staining for PECAM1/CD31 (green) of vascular endothelial cells in: vehicle controls (A), CTx (B), CTX + G-CSF/SCF (C), CTx + G-CSF (D). Immunofluorescent staining (PECAM1/CD31) was visualized using an epifluorescent microscope and a fluorescein isothiocyanate (FITC)/ tetramethyl rhodamine isothiocyanate (TRITC) filter cube, which helps distinguish specific fluorescence (green) from epifluorescence (red) and provides contrast to visualize tissue architecture. Scale bars = 10 μm. (E) Quantification of microvessel density (microvessels/mm2; mean ± SEM; n = 3 ovaries/group). Different letters above bars represent statistically significant differences (P<.05). CTx = cyclophosphamide/busulfan; CTx + G-CSF/SCF = cyclophosphamide/busulfan + granulocyte colony-stimulating factor/stem cell factor; CTx + G-CSF = cyclophosphamide/ busulfan + granulocyte colony-stimulating factor.

FIGURE 4

Granulocyte colony-stimulating factor ± stem cell factor (G-CSF ± SCF) treatment extends time to premature ovarian insufficiency in chemotherapy-treated female mice. (A) Percentage of female mice receiving vehicle (n = 5), cyclophosphamide and busulfan (CTx) (n = 7), CTx + G-CSF/SCF (n = 5), and CTx + G-CSF (n = 5) that produced litters in five successive breedings during a 6-month period. (B) Number of pups per litter during the course of the 6-month breeding trial in vehicle controls, CTx, CTx + G-CSF/SCF, and CTx + G-CSF alone. Asterisk denotes significant difference compared with vehicle controls (P<.05). (C) Average number of pups per breeding attempt across all five consecutive breedings. Values with different superscript letters are significantly different (P<.05). CTx = cyclophosphamide/busulfan; CTx + G-CSF = cyclophosphamide/busulfan + granulocyte colony-stimulating factor; CTx + G-CSF/SCF = cyclophosphamide/busulfan + granulocyte colony-stimulating factor/stem cell factor.