Loss of microRNA-7a2 induces hypogonadotropic hypogonadism and infertility - PubMed (original) (raw)

Loss of microRNA-7a2 induces hypogonadotropic hypogonadism and infertility

Kashan Ahmed et al. J Clin Invest. 2017.

Abstract

MicroRNAs (miRNAs) are negative modulators of gene expression that fine-tune numerous biological processes. miRNA loss-of-function rarely results in highly penetrant phenotypes, but rather, influences cellular responses to physiologic and pathophysiologic stresses. Here, we have reported that a single member of the evolutionarily conserved miR-7 family, miR-7a2, is essential for normal pituitary development and hypothalamic-pituitary-gonadal (HPG) function in adulthood. Genetic deletion of mir-7a2 causes infertility, with low levels of gonadotropic and sex steroid hormones, small testes or ovaries, impaired spermatogenesis, and lack of ovulation in male and female mice, respectively. We found that miR-7a2 is highly expressed in the pituitary, where it suppresses golgi glycoprotein 1 (GLG1) expression and downstream bone morphogenetic protein 4 (BMP4) signaling and also reduces expression of the prostaglandin F2a receptor negative regulator (PTGFRN), an inhibitor of prostaglandin signaling and follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion. Our results reveal that miR-7a2 critically regulates sexual maturation and reproductive function by interconnecting miR-7 genomic circuits that regulate FSH and LH synthesis and secretion through their effects on pituitary prostaglandin and BMP4 signaling.

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Conflict of interest statement

Conflict of interest: M. Stoffel is a member of the scientific advisory board of Regulus Therapeutics.

Figures

Figure 1

Figure 1. Ablation of mir-7a2 leads to male hypogonadism.

(A–C) Representative images of mir-7a2 WT (+/+) control and KO (–/–) testes (A) and quantification of testes weights (B) of mir-7a1 KO, mir-7a2 KO, or respective WT controls (mir-7a1 control, n = 7; mir-7a1 KO, n = 6; mir-7a2 control, n = 14; mir-7a2 KO, n = 9) at 6 to 8 weeks of age or (C) at 14 days (mir-7a2 control, n = 4; mir-7a2 KO, n = 2). Scale bar: 5 mm. (D and E) Immunohistological images (D) and quantification (E) of CYP17A1-positive Leydig cells (WT, mir-7a2 KO, n = 3). Scale bars: 50 μm. Arrows show Leydig cells. (F) Relative expression levels of steroidogenic genes in testes of mir-7a2 KO or control mice (WT, n = 9; mir-7a2 KO, n = 7). (G and H) Representative images of seminal vesicles (G) and quantification of weights (H) of control (upper) or mir-7a2 KO (lower) mice (WT, n = 6; mir-7a2 KO, n = 4). Scale bar: 5 mm. (I) Intratesticular testosterone levels normalized per total protein content in mir-7a2 KO or control mice (WT, n = 5; mir-7a2 KO, n = 3). (J) Total sperm count of the cauda epididymidis of 12-week-old mir-7a2 KO or control mice (WT, n = 5; mir-7a2 KO, n = 3). All data are represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA (B); t test (C, E, F, H, I, J).

Figure 2

Figure 2. Ablation of mir-7a2 leads to female hypogonadism and anovulation.

(A and B) Representative images of ovaries and uteri (A) and quantification of ovary weights (B) of mir-7a1 KO, mir-7a2 KO, or respective control mice (mir-7a1 control, n = 9; mir-7a1 KO, n = 10; mir-7a2 control, n = 7; mir-7a2 KO, n = 4). Scale bar: 5 mm. (C and D) Histological examination using H&E staining of ovaries of WT control (C) or mir-7a2 KO (D) mice (n = 4). CL, corpus luteum; LAF, large antral follicle; GF, growing follicle. Shown are representative images of 4 mice per genotype. Scale bar: 100 μm. (E) Relative expression levels of ovarian steroidogenic genes in mir-7a2 KO or control mice (WT, n = 6; mir-7a2 KO, n = 4). (F) Plasma estradiol levels of 8-week-old mir-7a2 KO or control mice (WT, n = 12; mir-7a2 KO, n = 11). All data are represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA (B); t test (E, F).

Figure 3

Figure 3. Constitutive genetic ablation of mir-7a2 causes hypogonadotropic hypogonadism.

(A) Expression of pituitary hormones in male mir-7a2 KO or control mice shown as heat map analysis from RNA-Seq (WT, mir-7a2 KO, n = 3). (B and C) Representative immunohistological images of pituitary sections (n = 3) stained for LH (B) or FSH (C) of control (upper images) or mir-7a2 KO mice (lower images). Scale bars: 200 μm. (D and E) Quantification of LH-positive (D) or FSH-positive (E) cells in pituitary of mir-7a2 KO or control mice (WT, mir-7a2 KO, n = 3). (F and G) Pituitary weights of male (F) or female (G) mir-7a1 KO, mir-7a2 KO, or respective control mice (males, mir-7a1 control, mir-7a1 KO, n = 4; mir-7a2 control, mir-7a2 KO, n = 8; mir-7a1 control, n = 8; mir-7a1 KO, n = 10; mir-7a2 control, n = 6; mir-7a2 KO, n = 7). (H and I) Plasma levels of FSH (H) or LH (I) in male mir-7a2 KO or control mice (WT, mir-7a2 KO, n = 7). (J and K) Plasma levels of FSH (J) or LH (K) in female mir-7a2 KO or control mice (WT, mir-7a2 KO, n = 4). (L) Number of oocytes collected after superovulation test in 5-week-old mir-7a2 KO or control mice (WT, n = 4; mir-7a2 KO, n = 3). All data are represented as mean ± SD except in (H, K), where data are represented as ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA (F, G); t test (D, E, H, I, J, K).

Figure 4

Figure 4. Acute ablation of mir-7a2 in adult mice recapitulates hypogonadotropic hypogonadism.

(A) TaqMan assays showing relative expression levels of miR-7a2, miR-7b, or miR-16 in pituitary of male UBC-Cre × mir-7a2fl/fl or Cre-negative mir-7a2fl/fl mice 10 weeks after a 5-day treatment with tamoxifen (TAM) or vehicle (UBC-Cre × mir-7a2fl/fl + vehicle, n = 5, mir-7a2fl/fl + TAM, UBC-Cre × mir-7a2fl/fl + TAM, n = 4). (B) Relative expression levels of pituitary hormones in UBC-Cre × mir-7a2fl/fl or mir-7a2fl/fl mice after treatment with tamoxifen or vehicle (UBC-Cre × mir-7a2fl/fl + vehicle, n = 5, mir-7a2fl/fl + TAM, UBC-Cre × mir-7a2flox + TAM, n = 4). (C and D) Immunohistological quantification of LH-positive (C) or FSH-positive (D) cells in pituitary sections of UBC-Cre × mir-7a2fl/fl or mir-7a2fl/fl mice after treatment with tamoxifen (mir-7a2fl/fl + TAM, n = 4; UBC-Cre × mir-7a2fl/fl + TAM, n = 3). (E and F) Plasma levels of FSH (E) or LH (F) of male UBC-Cre × mir-7a2fl/fl or mir-7a2fl/fl mice after treatment with tamoxifen or vehicle (FSH, UBC-Cre × mir-7a2fl/fl + vehicle, n = 5; mir-7a2fl/fl + TAM, UBC-Cre × mir-7a2fl/fl + TAM, n = 4; LH, UBC-Cre × mir-7a2fl/fl + vehicle, mir-7a2fl/fl + TAM, UBC-Cre × mir-7a2fl/fl + TAM, n = 3). (G) Testes weights of UBC-Cre × mir-7a2fl/fl or mir-7a2fl/fl mice after treatment with tamoxifen or vehicle (UBC-Cre × mir-7a2fl/fl + vehicle, n = 6; mir-7a2fl/fl + TAM, n = 10; UBC-Cre × mir-7a2fl/fl + TAM, n = 8). (H) Relative expression levels of the Leydig cell marker CYP17A1 in testes of UBC-Cre × mir-7a2fl/fl or mir-7a2fl/fl mice after treatment with tamoxifen or vehicle (UBC-Cre × mir-7a2fl/fl + vehicle, mir-7a2fl/fl + TAM, UBC-Cre × mir-7a2fl/fl + TAM, n = 4). All data are represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA (A, B, E, F, G, H); t test (C and D).

Figure 5

Figure 5. Pituitary gene expression analysis and identification of target genes of mir-7a2.

(A) Expression of key pituitary markers in mir-7a2 KO or control mice shown as heat map analysis from RNA-Seq of 6-week-old male mice (WT, mir-7a2 KO, n = 3). (B) Relative expression of key pituitary markers in UBC-Cre × mir-7a2fl/fl or Cre-negative mir-7a2fl/fl mice 10 weeks after a 5-day treatment with tamoxifen or vehicle (UBC-Cre × mir-7a2fl/fl + vehicle, n = 5, mir-7a2fl/fl + TAM, UBC-Cre × mir-7a2fl/fl +TAM, n = 4). (C) RNA-Seq results from pituitary of 6-week-old male mir-7a2 KO or control mice. Cumulative distributions of mRNA changes for predicted target genes of miR-7 (left)or miR-16 (right) with the indicated context+ score bins (color) or for genes with no respective miRNA site (black), as scored by the context+ model of TargetScan 6.2 (60). Number of genes per bin for miR-7: black, 11,884; green, 1,927; orange, 782; red, 245; purple, 83; miR-16: black, 12,841; green, 656; orange, 967; red, 306; purple, 151. (WT, mir-7a2 KO, n = 3). (D) Relative expression of predicted miR-7 target genes that were more than 1.3-fold upregulated in RNA-Seq in pituitaries of UBC-Cre × mir-7a2fl/fl or mir-7a2fl/fl mice 10 weeks after a 5-day treatment with tamoxifen relative to vehicle-treated mice (set as 1, black dotted line) (mir-7a2fl/fl + TAM, n = 4; UBC-Cre × mir-7a2fl/fl + TAM, n = 3). (E) Relative mRNA expression levels of miR-7 targets Glg1 and Ptgfrn in pituitary of WT control (mir-7a2+/+) or mir-7a2 KO (mir-7a2–/–) mice (n = 4). All data are represented as mean ± SD. *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001, ANOVA (B and C); t test (A, D, E).

Figure 6

Figure 6. miR-7a2 regulates gonadotropin production through BMP and prostaglandin signaling.

(A and B) Relative luciferase levels of plasmids carrying WT or mutated 3′ UTRs of Ptgfrn (A) or Glg1 (B) cotransfected in LbT2 cells with or without forced expression of mir-7a2 (n = 3). (C and D) Relative expression levels of gonadotropin genes in cells transfected with siRNA against Ptgfrn (C) or Glg1 (D) (n = 4). (E) Relative expression levels of gonadotropin genes, Cga, Fshb, and Lhb in cells overexpressing Ptgfrn (gray bars) or Glg1 (black bars) for 72 hours (n = 4). (F and G) Relative expression levels of Fshb (F) or Lhb (G) in LbT2 cells that were transfected with siPtgfrn or siCtrl and treated with 100 nM dinoprost or PBS for 4 hours (n = 4). (H) Concentration of LH in supernatants of cells 72 hours after silencing of Ptgfrn (n = 3). (I) Expression levels of Lhb in cells treated with dinoprost (100 nM), GREM1 (0.25 μg/ml), or dinoprost and GREM1 together (n = 3). (J) Western blot analysis of phospho-SMAD1/5/9 or total SMAD1 in lysates of cells pretreated with or without GREM1 (0.25 ug/ml) for 1 hour, followed by 30 minutes of stimulation with BMP4 (50 ng/ml). Shown is 1 representative experiment of 3. All data are represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA (E, F, G, I); t test (A, B, C, D, H).

Figure 7

Figure 7. Model illustrating the molecular pathways by which miR-7a regulates gonadotropic hormone secretion in the pituitary.

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References

    1. Abreu AP, Kaiser UB. Pubertal development and regulation. Lancet Diabetes Endocrinol. 2016;4(3):254–264. doi: 10.1016/S2213-8587(15)00418-0. - DOI - PMC - PubMed
    1. Herbison AE. Control of puberty onset and fertility by gonadotropin-releasing hormone neurons. Nat Rev Endocrinol. 2016;12(8):452–466. doi: 10.1038/nrendo.2016.70. - DOI - PubMed
    1. Leon S, Tena-Sempere M. Dissecting the roles of gonadotropin-inhibitory hormone in mammals: studies using pharmacological tools and genetically modified mouse models. Front Endocrinol (Lausanne) 2016;6:189. - PMC - PubMed
    1. Ramaswamy S, Weinbauer GF. Endocrine control of spermatogenesis: Role of FSH and LH/ testosterone. Spermatogenesis. 2015;4(2):e996025. - PMC - PubMed
    1. Christensen A, et al. Hormonal regulation of female reproduction. Horm Metab Res. 2012;44(8):587–591. doi: 10.1055/s-0032-1306301. - DOI - PMC - PubMed

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