Accelerated ovarian aging in mice by treatment of busulfan and cyclophosphamide

Abstract

Busulfan/cyclophosphamide (Bu/Cy) conditioning regimen has been widely used to treat cancer patients, while their effects on major internal organs in females are not fully understood. We treated female mice with Bu/Cy, and examined the histopathology of major internal organs on Day 30 after the treatment. The results show that Bu/Cy treatment affected the ovaries most extensively, while it had less effect on the spleen, lungs, and kidneys, and no effect on the heart, liver, stomach, and pancreas. To better understand the effect of Bu/Cy on the ovaries, we counted follicles, and determined the levels of ovarian steroids. The Bu/Cy-treated mice showed a reduction of primordial and primary follicles (P<0.01) on Day 30 and a marked loss of follicles at all developmental stages (P<0.01) on Day 60. Plasma levels of estradiol and progesterone in Bu/Cy-treated mice decreased by 43.9% and 61.4%, respectively. Thus, there was a gradual process of follicle loss and low estradiol in Bu/Cy-treated mice; this is a profile similar to what is found in women with premature ovarian failure (POF). The Bu/Cy-treated mice may serve as a useful animal model to study the dynamics of follicle loss in women undergoing POF.

Fig. 1

Effects of Bu/Cy treatment on histology of the major internal organs (a, b) The lungs; (c, d) The spleen; (e, f) The kidney; (g, h) The heart; (i, j) The stomach; (k, l) The liver; (m, n) The pancreas; (q, r) The ovary on Day 30; (s, t) The ovary on Day 60. H&E stainings of samples from Bu/Cy-treated mice were shown in (b), (d), (f), (h), (j), (l), (n), (r), and (t), and those from age-matched untreated mice were in (a), (c), (e), (g), (i), (k), (m), (q), and (s). There were thinner uterine walls and vaginal walls in Bu/Cy-treated mice (o, right) compared with the age-matched controls (o, left). The ovary of Bu/Cy-treated mice (p, right) was smaller than that of the controls (p, left). There was a significant reduction of primordial and primary follicles in Bu/Cy-treated mice (r) relative to age-matched untreated controls (q); There was a marked loss of follicles and mild fibrosis in part of interstitial area (arrow) in Bu/Cy-treated mice (t) compared with the controls (s)

Fig. 2

Effect of Bu/Cy treatment on different follicle types On Day 30, there was a significant reduction (P<0.01) of primordial and primary follicles in Bu/Cy-treated mice (hatched bar) compared with age-matched untreated mice (black bar). On Day 60, there was a significant reduction (P<0.01) of primordial, primary, secondary, and antral follicles in Bu/Cy-treated mice (white bar) compared with the controls (black bar). Each bar represents the mean±standard error of the mean (SEM) (n=5). ^** P<0.01 compared with untreated female mice at the age of three months

Fig. 3

Plasma levels of estradiol (a), progesterone (b), and testosterone (c) in mice Levels of estradiol and progesterone were tested on Day 30. (a) The plasma level of estradiol in Bu/Cy-treated mice (white bar) decreased by 43.9% compared with the control (black bar). (b) The plasma level of progesterone in Bu/Cy-treated mice (white bar) decreased by 61.4% compared with the control (black bar). (c) There were no significant changes of testosterone levels between Bu/Cy-treated mice and the control. Values are the mean±SEM (n=6). ^** P<0.01 compared with untreated age-matched female mice. Control represents the age-matched untreated mice. Treated represents the Bu/Cy-treated mice