Sexually dimorphic mammalian tissues, including sexual organs and the brain, contain stem cells that are directly or indirectly regulated by sex hormones1,2,3,4,5,6. 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 hormones7,8,9,10, 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.
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S.J.M. is a Howard Hughes Medical Institute Investigator and the Mary McDermott Cook Chair in Pediatric Genetics. This work was supported by the Cancer Prevention and Research Institute of Texas (awards to D.N. and S.J.M.) and the National Heart Lung and Blood Institute (HL097760 to S.J.M.). B.P.L. was supported by an Irvington Institute-Cancer Research Institute/Edmond J. Safra Memorial Fellowship. Flow-cytometry was partially supported by the National Institutes of Health (NCRR grant S10RR024574, NIAID AI036211 and NCI P30CA125123) for the BCM Cytometry and Cell Sorting Core. We also thank J. Richards, S. Mani and former members of the Nakada laboratory for discussions. This work was initiated in the Life Sciences Institute at the University of Michigan then completed at Baylor College of Medicine and Children’s Research Institute at UT Southwestern. We thank the University of Virginia Center for Research in Reproduction for measuring serum hormone levels. This work is dedicated to Nicole Ryan who passed away during the study.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Castration or ovariectomy modestly increased the numbers of B cells in the bone marrow without affecting the numbers of haematopoietic stem cells or MPPs.
a, Castration (cast) or ovariectomy (ovx) did not significantly affect the numbers of haematopoietic stem cells or MPPs in the bone marrow (femurs and tibias). b, Castration or ovariectomy significantly increased the numbers of B220+ B cells in the bone marrow but did not affect the numbers of Mac1+/Gr1+ myeloid cells, CD3+ T cells, or Ter119+ erythroid cells in the bone marrow or spleen. 3 sham and 4 gonadectomized mice used in 3 independent experiments. All data represent mean ± standard deviation; *P < 0.05 by Student’s t-test.
Extended Data Figure 2 Administration of oestradiol (E2) to mice induced erythropoiesis in the spleen.
a–c, Treatment of male and female mice for 1 week with E2, with or without P, did not affect the numbers of Mac1+/Gr1+ myeloid cells, B220+ B cells, or CD3+ T cells in the bone marrow or spleen of either sex. d, E2 and E2+P treatment did significantly increase the number of Ter119+ erythroid cells in the spleen of male mice, and E2 treatment significantly increased the number of Ter119+ erythroid cells in the spleen of female mice. n = 3 mice per treatment in 3 independent experiments. All data represent mean ± standard deviation; *P < 0.05; **P < 0.005; ***P < 0.0005 by Student’s t-test comparing each treatment to vehicle.
Extended Data Figure 3 Administration of exogenous oestrogen and progesterone significantly increased serum oestrogen and progesterone levels in mice but progesterone did not affect haematopoietic stem-cell division in vivo.
a, Oestradiol treatment significantly increased serum oestradiol levels in male and female mice but the increased levels remained within the physiological range, similar to levels observed during pregnancy (see Fig. 4e) (male oil, 22; male E2, 20; female oil, 33; female E2, 14 mice used in 8 independent experiments). b, Progesterone treatment significantly increased serum progesterone levels in male and female mice (n = 3 mice per treatment in 3 independent experiments). Note that this did not affect bone marrow or spleen cellularity, haematopoietic stem-cell frequency, or haematopoietic stem-cell division (Fig. 2b–d). c, Esr1-deficient mice had normal levels of serum progesterone (n = 3 mice per group in 3 independent experiments). d–f, Administration of a progesterone receptor antagonist, RU486 (RU), did not affect bone marrow or spleen cellularity (d), haematopoietic stem-cell frequency in the bone marrow (e), or the division of haematopoietic stem cells, MPPs, or WBM cells (f). All data represent mean ± standard deviation from 3 independent experiments, except as indicated above; *P < 0.05; **P < 0.005; ***P < 0.0005 by Student’s t-test comparing each treatment to vehicle (oil).
Extended Data Figure 4 Oestrogen treatment increased the frequency of Ki67+ cycling haematopoietic stem cells.
a, Administration of oestrogen (E2) significantly increased the frequency of haematopoietic stem cells in G1 phase of the cell cycle, and reduced the frequency of haematopoietic stem cells in G0 phase of the cell cycle based on Ki67/propidium iodide staining (n = 3 mice per treatment in 3 independent experiments). b, c, Col1A1-H2B-GFP; Rosa26-M2-rtTA mice pulsed with doxycycline for 6 weeks to induce H2B–GFP expression were treated with oil (blue histogram) or E2 (red histogram) for 2 weeks without doxycycline. E2 treatment significantly increased the rate of haematopoietic stem-cell division as indicated by the reduced frequency of GFPhigh quiescent haematopoietic stem cells and the increased frequency of GFPlow moderately cycling haematopoietic stem cells (5 oil-treated and 4 E2-treated mice used in 3 independent experiments). All data represent mean ± standard deviation; *P < 0.05 by Student’s t-test comparing each treatment to vehicle (oil).
Extended Data Figure 5 Female mice have increased frequencies of megakaryocyte–erythroid progenitors (MEPs) and apoptotic Ter119+ cells relative to male mice.
a, Annexin-V staining of the indicated cell populations in the bone marrow of male and female mice revealed a significantly increased frequency of apoptotic Annexin-V+ cells among Ter119+ erythroid progenitors in female mice. b, Female mice had a significantly increased frequency of CD34−CD16/32−Lin−Sca-1−c-kit+ MEPs, but no significant differences in the frequencies of CD34+CD16/32−Lin−Sca-1−c-kit+ CMPs, CD34+CD16/32+Lin−Sca-1−c-kit+ GMPs, or Flt3+IL-7R+Lin−Sca-1lowc-kitlow CLPs. c, None of the restricted progenitors or differentiated cells displayed differences in cell-cycle status between male and female mice (a–c, n = 5 mice per group in three independent experiments). Data represent mean ± standard deviation; *P < 0.05; **P < 0.005; by Student’s t-test comparing each treatment between sexes.
Extended Data Figure 6 Inhibiting oestrogen signalling by anastrozole treatment or Esr1 deficiency did not affect the numbers of haematopoietic cells in the bone marrow or spleen.
a, Administration of anastrozole (Ana) to mice for 2 weeks did not significantly affect the number of Ter119+ erythroid cells, CD3+ T cells, B220+ B cells, or Mac1+/Gr1+ myeloid cells in the bone marrow or spleen (4 PBS-treated and 6 anastrozole-treated mice were used in 4 independent experiments). b, c, Esr1 deficiency did not significantly affect bone marrow cellularity (b) or the frequencies of haematopoietic stem cells or MPPs (c) in either sex. d, Esr1 deficiency did not significantly affect the numbers of Ter119+ erythroid cells, CD3+ T cells, B220+ B cells, or Mac1+/Gr1+ myeloid cells in the bone marrow or spleen of normal mice. −/− indicates Esr1-deficient mice and +/+ indicates wild-type littermate control mice (b–d, n = 3 mice per group in 3 independent experiments). All data represent mean ± standard deviation.
Extended Data Figure 7 Pharmacological ERα activation, but not ERβ activation, is sufficient to promote haematopoietic stem-cell division.
Male mice (n = 5 mice per treatment in 3 independent experiments) were treated with oil, ERα agonist PPT, or ERβ agonist DPN for 14 days then pulsed with BrdU for 10 days, beginning on the fourth day of hormone treatment. a, b, PPT or DPN treatment did not significantly affect the cellularity of bone marrow or spleen (a), or the frequencies of haematopoietic stem cells or MPPs in bone marrow (b). c, PPT treatment, but not DPN treatment, significantly increased erythropoiesis in bone marrow and spleen. d, PPT significantly increased the division rates of haematopoietic stem cells and MPPs, but DPN failed to do so, suggesting that ERα activation, but not ERβ activation, promotes haematopoietic stem cell division. Data represent mean ± standard deviation; ***P < 0.0005 by Student’s t-test comparing each treatment to vehicle (oil).
Extended Data Figure 8 E2 treatment changes haematopoietic stem-cell gene expression profile in a manner consistent with increased cell division.
a, b, Gene set enrichment analysis revealed that haematopoietic stem cells from mice treated with E2 exhibited significant enrichment in the expression of genes involved in cell cycling (a). We also observed a significant enrichment in the expression of genes with E2F1 motifs in haematopoietic stem cells from E2-treated mice, consistent with the role of E2Fs in cell-cycle control (b). n = 3 mice per treatment of each sex were used to isolate independent aliquots of RNA from haematopoietic stem cells for gene expression profiling.
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Nakada, D., Oguro, H., Levi, B. et al. Oestrogen increases haematopoietic stem-cell self-renewal in females and during pregnancy. Nature 505, 555–558 (2014). https://doi.org/10.1038/nature12932
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