Oestrogen increases haematopoietic stem-cell self-renewal in females and during pregnancy

Abstract

Sexually dimorphic mammalian tissues, including sexual organs and the brain, contain stem cells that are directly or indirectly regulated by sex hormones. An important question is whether stem cells also exhibit sex differences in physiological function and hormonal regulation in tissues that do not show sex-specific morphological differences. The terminal differentiation and function of some haematopoietic cells are regulated by sex hormones, but haematopoietic stem-cell function is thought to be similar in both sexes. Here we show that mouse haematopoietic stem cells exhibit sex differences in cell-cycle regulation by oestrogen. Haematopoietic stem cells in female mice divide significantly more frequently than in male mice. This difference depends on the ovaries but not the testes. Administration of oestradiol, a hormone produced mainly in the ovaries, increased haematopoietic stem-cell division in males and females. Oestrogen levels increased during pregnancy, increasing haematopoietic stem-cell division, haematopoietic stem-cell frequency, cellularity, and erythropoiesis in the spleen. Haematopoietic stem cells expressed high levels of oestrogen receptor-α (ERα). Conditional deletion of ERα from haematopoietic stem cells reduced haematopoietic stem-cell division in female, but not male, mice and attenuated the increases in haematopoietic stem-cell division, haematopoietic stem-cell frequency, and erythropoiesis during pregnancy. Oestrogen/ERα signalling promotes haematopoietic stem-cell self-renewal, expanding splenic haematopoietic stem cells and erythropoiesis during pregnancy.

Figure 1. HSCs divide more frequently in female as compared to male mice

a-c, The frequency of HSCs and MPPs in the bone marrow (a), the total numbers of HSCs and MPPs in two femurs and tibias (b), and the numbers of HSCs and MPPs per gram of body mass (c) did not differ between young adult male and female mice. d, BrdU incorporation into whole bone marrow (WBM) cells, HSCs, and MPPs during a 10 day pulse (a-d, n=5 mice/group in 5 independent experiments). e, H2B-GFP intensity immediately after a 6-week pulse of doxycycline in Col1A1-H2B-GFP; Rosa26-M2-rtTA mice (left) or after a 12-week chase without doxycycline (right). f, The percentages of WBM cells, HSCs, and MPPs that retained H2B-GFP (4 males and 3 females in 3 independent experiments). All data represent mean±standard deviation; *, p<0.05; **, p<0.005; and ***, p<0.0005 by Student’s t-test.

Figure 2. Increased HSC division in female mice depends upon the ovaries and is stimulated by estradiol

a, Effect of castration or ovariectomy on the rates of division by WBM cells, HSCs, or MPPs (3 sham and 4 gonadectomized mice in 3 independent experiments). b, c, Administering estradiol (E2), progesterone (P), or both (E2+P) for 1 week did not affect the number of bone marrow cells or splenocytes (b), or HSC frequency in bone marrow (c). d, Administering E2 or E2+P significantly increased HSC division in male and female mice (b-d, n=3 mice/treatment in 3 independent experiments). e, f, Administering E2 to castrated or ovariectomized mice significantly increased HSC division by BrdU incorporation (e, n=5) or Ki-67 staining (f, n=3). g,h, Administering dihydrotestosterone (DHT) for 7 days did not affect HSC division or HSC frequency (n=4 mice/treatment in 4 independent experiments). Data represent mean±standard deviation; *, p<0.05; **, p<0.005; and ***, p<0.0005 by Student’s t-test.

Figure 3. Estradiol-ERα signaling promotes HSC division in female mice

a, b, Bone marrow cellularity (a), HSC, and MPP frequency (b) in mice administered the aromatase inhibitor anastrozole (A) or vehicle (PBS, abbreviated V) for 2 weeks. c, BrdU incorporation (10 day pulse) by WBM cells, HSCs, or MPPs in male or female mice treated with anastrozole or vehicle (a-c, 4 PBS treated and 6 anastrozole treated mice in 4 independent experiments). d, qRT-PCR revealed that HSCs and MPPs from female and male mice expressed greatly elevated levels of Esr1 (which encodes ERα) but not Esr2 (ERβ), Pgr (progesterone receptor), or Ar (androgen receptor) relative to male WBM (*, p<0.05 between HSC/MPP and WBM; #, p<0.05 between male and female.). Expression levels were normalized to β-actin. e, Immunofluorescence for ERα in HSCs (d,e, n=3 mice from 3 experiments. Scale bar, 4 μm). f, BrdU incorporation (10-day pulse) by WBM cells, HSCs, and MPPs in male and female mice (−/−, Esr1-deficient; +/+, littermate controls, f-h, n=3 mice/group in 3 independent experiments). g, Conditional deletion of Esr1 in female Mx1-Cre; Esr1^fl/fl mice reduced BrdU incorporation into HSCs (Cre⁻, Esr1^fl/fl; Mx1, Mx1-Cre; Esr1^fl/fl, n=3). h, i, Conditional deletion of Esr1 in male Mx1-Cre; Esr1^fl/fl mice (h) or Vav1-Cre; Esr1^fl/fl mice (i) rendered HSCs insensitive to exogenous estrogen (h, i, n=3 mice/group in 2 independent experiments). j, E2 treatment of mice competitively reconstituted with WBM cells from wild-type and Vav1-Cre; Esr1^fl/fl mice increased BrdU incorporation by wild-type HSCs but not Esr1-deficient HSCs (3 oil-treated and 4 E2-treated mice in 2 independent experiments). k, Effect of E2 on HSCs freshly added to culture (serum-free, phenol red-free medium with E2 or vehicle for three days; BrdU for one hour; n=3 mice in 2 independent experiments). All data represent mean±standard deviation; * and #, p<0.05; ** and ##, p<0.005; and *** and ###, p<0.0005 by Student’s t-test.

Figure 4. Increased HSC division, HSC frequency, and erythropoiesis in the spleen during pregnancy depend upon ERα signaling in haematopoietic cells

a, Spleen and bone marrow cellularity. Pregnant mice were between days 12 and 15 of gestation. b, Pregnant mice had significantly increased Mac-1/Gr-1⁺ myeloid cells, Ter119⁺ erythroid cells, and overall cellularity in the spleen, but reduced bone marrow B220⁺ B-cells. The increase in splenic erythropoiesis required ERα expression by haematopoietic cells. c, HSC frequency in the bone marrow and spleen. d. In pregnant mice the rate of BrdU incorporation (24-hour pulse) significantly increased in whole bone marrow (WBM) cells, bone marrow HSCs, and spleen HSCs and depended upon ERα expression by haematopoietic cells (a-d, 9 non-pregnant, 7 pregnant Esr1^fl/fl, and 6 pregnant Mx1-Cre; Esr1^fl/fl mice in 9 independent experiments). e, Serum E2 levels in mice (21 non-pregnant and 9 pregnant mice from 6 independent experiments). All data represent mean±standard deviation; *, p<0.05; **, p<0.005; and ***, p<0.0005 by Student’s t-test.