Research ArticleENDOMETRIOSIS

Loss of HDAC3 results in nonreceptive endometrium and female infertility

See allHide authors and affiliations

Science Translational Medicine  09 Jan 2019:
Vol. 11, Issue 474, eaaf7533
DOI: 10.1126/scitranslmed.aaf7533
  • Fig. 1 Attenuation of HDAC3 in the eutopic endometrium from women with endometriosis as well as baboon and mouse models.

    (A) Immunohistochemical H-score and representative photomicrographs of HDAC3 proteins in the proliferative, early secretory, and mid-secretory phase endometrium from women without and with endometriosis (n = 7 per phase for each group). (B) Immunohistochemical H-score in stroma and representative photomicrographs of HDAC3 in the endometriosis baboon model. Endometriosis was induced by intraperitoneal inoculation of menstrual endometrium. The expression of HDAC3 was examined in the baboon endometrium before inoculation and 3, 9, and 15 months after inoculation (n = 4). (C) Immunohistochemical H-score and representative photomicrographs of HDAC3 in the eutopic endometrium from the endometriosis mouse model. Endometriosis was surgically induced in mice, and then the expression of HDAC3 was examined by immunohistochemical analysis (n = 6). Nuclei were counterstained with hematoxylin (blue). Mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test for data with only two groups and ANOVA followed by Tukey or Bonferroni test for pairwise t test for data containing more than two groups. Scale bars, 50 μm. Ctrl, control; Eosis, endometriosis; Prolif, proliferative phase; Sec, secretory phase; IgG, immunoglobulin G.

  • Fig. 2 Defects of implantation and decidualization in Hdac3d/d mice.

    (A) Hdac3 ablation in the uteri of Hdac3d/d mice. RT-qPCR analysis of Hdac3 gene expression in the uterus of Hdac3f/f and Hdac3d/d mice at 2 months of age (n = 6 for each genotype). Western blot analysis of HDAC1, HDAC2, and HDAC3 proteins in the uteri of Hdac3f/f and Hdac3d/d mice at 2 months of age. Actin was used as sample loading control. Representative images of immunohistochemical staining for HDAC3 in the uteri of Hdac3f/f and Hdac3d/d mice at 2 months of age. Scale bars, 50 μm. (B) Implantation sites in uteri of Hdac3f/f and Hdac3d/d mice at GD 5.5. Scale bars, 1 cm. Representative images of hematoxylin and eosin (H&E) staining in uteri of Hdac3f/f and Hdac3d/d mice at GD 5.5. Arrowheads indicate embryos. Scale bars, 200 μm. (C) Ratio of uterine weight to body weight and representative photographs of uteri from Hdac3f/f (n = 6) and Hdac3d/d (n = 4) mice after artificially induced decidualization. Unfilled arrowheads indicate stimulated horns. Scale bars, 1 cm. (D) Percentages and representative photomicrographs of immunohistochemical staining for Ki-67 (brown), a proliferation marker, in endometrial epithelial and stromal cells from Hdac3f/f and Hdac3d/d mice at GD 3.5 (n = 6 for each genotype). Nuclei were counterstained with hematoxylin (blue). Scale bars, 50 μm. Mean ± SEM. ***P < 0.001, Student’s t test.

  • Fig. 3 Decreased progesterone signaling in the uteri of Hdac3d/d mice.

    (A) Immunohistochemical H-score and representative photomicrographs of ESR1 and PGR in stromal cells in the endometrium from Hdac3f/f and Hdac3d/d mice at GD 3.5 (n = 6 for each genotype). Scale bars, 100 μm. (B) RT-qPCR analysis of expression of progesterone target genes Lrp2, Fst, Areg, and Il13ra2 in the uteri of Hdac3f/f and Hdac3d/d mice at GD 3.5 (n = 6 for each genotype). (C) Percentages and representative photomicrographs of vimentin in stromal cells in the endometrium from Hdac3f/f and Hdac3d/d mice at GD 3.5 (n = 6 for each genotype). Scale bars, 25 μm. (D) Percentages and representative photomicrographs of COUP-TFII in stromal cells in the endometrium from Hdac3f/f and Hdac3d/d mice at GD 3.5 (n = 6 for each genotype). Nuclei were counterstained with hematoxylin (blue). Scale bars, 25 μm. Mean ± SEM. * P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test.

  • Fig. 4 Overexpression of collagen in the endometrium of mice and women with endometriosis.

    (A) Masson’s trichrome staining (MTS) in the endometriosis mouse model. (B) Intensity of Masson’s trichrome staining and representative photomicrographs of the endometrium from women with and without diagnosed endometriosis (n = 6 for each group per phase). Scale bars, 25 μm. (C) Immunohistochemical H-score and representative photomicrographs of COL1 in the eutopic endometrium from women with and without endometriosis (n = 7 for each group per phase) at proliferative, early secretory, and mid-secretory phases. Nuclei were counterstained with hematoxylin (blue). Scale bars, 50 μm. Mean ± SEM. *P < 0.05 and ***P < 0.001, Student’s t test.

  • Fig. 5 The effect of HDAC3 knockdown on decidualization in hESCs.

    (A) Western blot analysis of HDAC3 in hESCs from infertile women with (n = 5) or without (n = 6) endometriosis. (B) RT-qPCR analysis of expression of decidualization marker genes, IGFBP1 and PRL, during in vitro decidualization of hESCs treated with nontargeting negative control siRNA (N.C. siRNA) or HDAC3 siRNA (n = 6). (C) Western blot analysis of PGR during in vitro decidualization of hESCs treated with negative control siRNA or HDAC3 siRNA in OPTI-MEM medium containing 10 nM estradiol, 1 mM medroxyprogesterone acetate, and 50 μM cAMP (EPC). (D) Knockdown of HDAC3 induces derepression of both COL1A1 and COL1A2 mRNA expression. Human stromal cells were transfected with negative control siRNA or HDAC3 siRNA and cultured for the indicated duration. The expression of indicated genes was analyzed by RT-qPCR (n = 6). Mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, ANOVA followed by Tukey or Bonferroni test for pairwise t test for data containing more than two groups.

  • Fig. 6 The effect of HDAC3 knockdown on the transcriptional regulation of collagen genes in hESCs.

    (A) HDAC3 knockdown enhances the selective recruitment of p300 to the HDAC3-BE of collagen genes. hESCs were transfected with nontargeting negative control siRNA (N.C. siRNA) or HDAC3 siRNA. ChIP assay was performed with the indicated antibodies. DNA samples were analyzed by qPCR (n = 3). (B) Recruitment of p300 to the HDAC3-BE is increased by Hdac3 ablation in mouse uteri. ChIP assay was performed using the indicated antibodies. DNA samples were analyzed by qPCR (n = 3). (C) p300 knockdown represses the transcriptional activation of collagen genes through knockdown of HDAC3. hESCs were transfected with negative control siRNA or HDAC3 siRNA and then treated with C646, a p300 inhibitor, or transfected with negative control siRNA or p300 siRNA. ChIP assays were performed with the indicated antibodies. DNA samples were analyzed by qPCR (n = 3). (D) The treatment of hESCs with C646 or p300 siRNA inhibits the transcriptional activation of both COL1A1 and COL1A2 genes during in vitro decidualization of hESCs with HDAC3 siRNA. hESCs were transfected with negative control siRNA or HDAC3 siRNA and then treated with C646 or transfected with negative control siRNA or p300 siRNA. The expression of the indicated genes was analyzed by RT-qPCR (n = 3). (E) Inhibition of p300 function reversed the decreased transcription of decidualization marker genes, IGFBP1 and PRL, upon HDAC3 knockdown during in vitro decidualization of hESCs. hESCs were transfected with negative control siRNA or HDAC3 siRNA and then treated with C646 or transfected with negative control siRNA or p300 siRNA. The expression of indicated genes was analyzed by RT-qPCR (n = 3). Mean ± SEM. *P < 0.05 and **P < 0.01, ANOVA followed by Tukey or Bonferroni test for pairwise t test for data containing more than two groups.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/474/eaaf7533/DC1

    Fig. S1. The expression of HDAC3 in mouse uteri during early pregnancy and in all phases of the estrous cycle.

    Fig. S2. Normal ovarian histology and serum progesterone and estrogen in Hdac3d/d mice.

    Fig. S3. Decidualization defect in Hdac3d/d mice after artificially induced decidualization.

    Fig. S4. Increased collagen in the uteri of Hdac3d/d mice.

    Fig. S5. Motif analysis on HDAC3 interval sequences.

    Fig. S6. Identification of Hdac3 target genes in the mouse uterus.

    Fig. S7. Recruitment of HDAC3 on COL1A1 and COL1A2 genes.

    Fig. S8. Overexpression of collagen in the uteri of Hdac3d/d mice.

    Fig. S9. Immunohistochemical analysis of COL1 in the uteri of Hdac3d/d mice.

    Table S1. Infertility in Hdac3d/d female mice.

    Table S2. Normal ovulation and fertilization in Hdac3d/d female mice.

    Table S3. Dysregulation of atherosclerosis and fibrosis pathways in the uteri of Hdac3d/d mice.

    Table S4. List of primers used in RT-qPCR analysis.

    Table S5. Raw data (provided as an Excel file).

  • The PDF file includes:

    • Fig. S1. The expression of HDAC3 in mouse uteri during early pregnancy and in all phases of the estrous cycle.
    • Fig. S2. Normal ovarian histology and serum progesterone and estrogen in Hdac3d/d mice.
    • Fig. S3. Decidualization defect in Hdac3d/d mice after artificially induced decidualization.
    • Fig. S4. Increased collagen in the uteri of Hdac3d/d mice.
    • Fig. S5. Motif analysis on HDAC3 interval sequences.
    • Fig. S6. Identification of Hdac3 target genes in the mouse uterus.
    • Fig. S7. Recruitment of HDAC3 on COL1A1 and COL1A2 genes.
    • Fig. S8. Overexpression of collagen in the uteri of Hdac3d/d mice.
    • Fig. S9. Immunohistochemical analysis of COL1 in the uteri of Hdac3d/d mice.
    • Table S1. Infertility in Hdac3d/d female mice.
    • Table S2. Normal ovulation and fertilization in Hdac3d/d female mice.
    • Table S3. Dysregulation of atherosclerosis and fibrosis pathways in the uteri of Hdac3d/d mice.
    • Table S4. List of primers used in RT-qPCR analysis.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5. Raw data (provided as an Excel file).

Stay Connected to Science Translational Medicine

Navigate This Article