Young adult donor bone marrow infusions into female mice postpone age-related reproductive failure and improve offspring survival

Abstract

The female reproductive axis is the first major organ system of the body to fail with advancing age. In addition to a permanent cessation of fertile potential, the loss of cyclic ovarian function in humans heralds the onset of menopause, which in turn underlies the emergence of a diverse spectrum of health issues in aging women. Recently, it was reported that bone marrow (BM) transplantation (BMT) into adult female mice conditioned a week earlier with highly cytotoxic drugs rescues ovarian function and fertility. Herein we show in mice receiving no prior conditioning regimen that once-monthly infusions of BM-derived cells retrieved from young adult female donors bearing an enhanced green fluorescent protein (EGFP) transgene sustain the fertile potential of aging wild-type females long past their time of normal reproductive senescence. The fertility-promoting effects of female donor BM are observed regardless whether the infusions are initiated in young adult or middle-aged females. Although the mechanism by which BM infusions benefit the reproductive performance of aging females remains to be elucidated, the absence of EGFP-expressing offspring suggests that it does not depend on development of mature eggs derived from germline-committed cells in the donor marrow. However, donor BM-derived somatic cells accumulate in the recipients, indicating efficient donor cell engraftment without prior conditioning. These findings provide a strong impetus to further explore development of adult stem cell-based technologies to safely extend function of the female reproductive axis into advanced age without the need for toxic pre-conditioning protocols routinely used in other models of stem cell delivery.

Keywords: aging; bone marrow; fertility; menopause; ovary; reproduction; stem cell.

Figure 1.. Repeated BM-INF delay female reproductive aging.

(A) Number of full-term pregnancies achieved by female mice at the indicated ages following once-monthly infusions of vehicle (VEH-INF) or BM harvested from young adult female donors (BM-INF), initiated at 3 months of age. The total number of recipients analyzed in each age bracket is indicated in parentheses over the respective bars. (B) Percentage of VEH-INF and BM-INF females that achieved full-term pregnancies between 8.5-11.5, 11.5-14.5 and 14.5-17.5 months of age, as calculated from the raw data shown in panel A.

Figure 2.. Reproductive performance of aging female mice after once-monthly infusions of vehicle or BM harvested from young adult female donors, initiated at 8 months of age.

Fertility outcomes are shown for each VEH-INF female and BM-INF female between 10-11.5 (A), 11.5-13 (B), 13-14.5 (C), 14.5-16 (D), and 16-17.5 (E) months (M) of age (each mouse is represented by a number on the x-axis) run in parallel mating trials. The total number of offspring delivered and that survived for each female are indicated. Crosses designate mice that had to be euthanized due to severe health complications or that died of natural causes during the study period.

Figure 3.. Pooled analysis of the effects of BM-INF on reproductive function in aging females.

Percentage of VEH-INF and BM-INF females that achieved full-term pregnancies at the indicated ages, as calculated from the combined raw data shown in figure 1A (infusions started at 3 months of age) and figure 2 (infusions started at 8 months of age). A significantly (Fisher's exact test) higher percentage of BM-INF females achieved full-term pregnancies between 11.5-14.5 and 14.5-17.5 months of age compared with age-matched VEH-INF females.

Figure 4.. Repeated BM-INF do not affect fecundity but dramatically improve survival rates of offspring delivered by aging females.

Summary of fecundity (mean ±SEM) of female mice that achieved full-term pregnancies at the indicated ages following once-monthly infusions of vehicle (VEH-INF) or BM harvested from young adult female donors (BM-INF), starting at 3 months (A) or 8 months (B) of age. (C) Offspring number and survival rates in mating trials of VEH-INF or BM-INF females between 10-17.5 months of age following once-monthly infusions initiated at 8 months of age. In addition to a marked increase in the total number of offspring delivered, the number of offspring delivered that survived was significantly increased by BM-INF (*, P = 0.0001 versus the VEH-INF group by Fisher's exact test).

Figure 5.. Analysis of donor cell engraftment in recipients after BM-INF without prior conditioning.

Representative immunohistochemical analysis of EGFP expression (brown) in ovaries of aged wild-type females following 15 once-monthly infusions of BM harvested from young adult β-actin-EGFP transgenic female donors (A, B). The ovary of a representative transgenic donor female (C) is shown as a positive control. (D) Chimerism analysis of BM-derived cells collected from female mice following 15 once-monthly infusions of β-actin -EGFP transgenic BM (BM-INF, X15) or a single infusion of β-actin-EGFP transgenic BM (BM-INF, X1). Parallel analysis of BM harvested from recipient females conditioned with busulfan and cyclophosphamide prior to BMT (again using β transgenic females as donors) is shown for comparison. For these samples, BM was collected 2 weeks (wk) and 4 weeks post-BMT. The data shown represent the mean ± SEM of results from analysis of 4 mice per group.

Figure 6.. Male donor BM does not replicate the pro-fertility effects of female donor BM.

Percentage of female mice receiving vehicle (Control, n = 18), a combination chemotherapy regimen containing busulfan and cyclophosphamide (CTx, n = 21) or CTx followed by bone marrow transplantation 1 week later, using young adult male (MBMT, n = 16) or female (FBMT, n = 19) donors, that achieved full-term pregnancies over a subsequent 7-month period when mating was initiated coincident with the transplants. Data shown are the mean ± SEM of combined results from 3 separate trials (*, P < 0.05 versus the respective CTx group or CTx + MBMT group).