Increased gene copy number of VAMP7 disrupts human male urogenital development through altered estrogen action

Journal name:
Nature Medicine
Volume:
20,
Pages:
715–724
Year published:
DOI:
doi:10.1038/nm.3580
Received
Accepted
Published online

Abstract

Despite the fact that genitourinary defects are among the most common birth defects in newborns, little is known about their etiology. Here we analyzed children born with congenital genitourinary tract masculinization disorders by array-comparative genomic hybridization, which revealed in 1.35% of cases the presence of de novo copy number gains at Xq28 encompassing the VAMP7 gene, which encodes a vesicle-trafficking protein that is part of the SNARE complex. Transgenic mice carrying a bacterial artificial chromosome encoding human VAMP7 mimicked the defective urogenital traits observed in boys with masculinization disorders such as cryptorchidism, urethral defects and hypospadias. Transgenic mice also exhibited reduced penile length, focal spermatogenic anomalies, diminished sperm motility and subfertility. VAMP7 colocalized with estrogen receptor α (ESR1) in the presence of its cognate ligand, 17β-estradiol. Elevated levels of VAMP7 markedly intensified ESR1-potentiated transcriptional activity by increasing ESR1 protein cellular content upon ligand stimulation and upregulated the expression of estrogen-responsive genes including ATF3, CYR61 and CTGF, all of which have been implicated in human hypospadias. Hence, increased gene dosage of VAMP7, and thus higher expression levels of its protein product, enhances estrogen receptor action in male genitourinary tissues, affects the virilization of the reproductive tract and results in genitourinary birth defects in humans.

At a glance

Figures

  1. A terminal Xq28 gain encompassing VAMP7 in 46,XY children presenting with masculinization disorders of the urogenital tract.
    Figure 1: A terminal Xq28 gain encompassing VAMP7 in 46,XY children presenting with masculinization disorders of the urogenital tract.

    (ab) Genomic hybridization profiles of unrelated 46,XY subjects with cryptorchidism (a) or hypospadias (b) compared with a gender-matched reference DNA. The mean log2 ratio (patient/reference) of intensity for each single probe is presented across individual chromosomes. Red arrows indicate a terminal X copy gain (green). (c) Representative FISH hybridization of phytohemagglutinin-stimulated blood lymphocytes from subjects with duplication of Xq28 detected by CGH array using RP11-479B17 probe (red) specific to the pseudoautosomal region 2, including Xq28 and Yq12 loci. RP11-815E21 at Xq22.3 (green) was used as a hybridization control. White arrow indicates copy number gain. Scale bar, 5 μm. Data are representative of three independent experiments. (d) Literature review of individuals with Xq28 duplication encompassing VAMP7 and presenting with defective virilization of the male reproductive tract. For references, see Supplementary Table 1.

  2. VAMP7 is present in human and mouse male reproductive tissues.
    Figure 2: VAMP7 is present in human and mouse male reproductive tissues.

    (a) Western blot analysis of VAMP7 expression in human fetal testes and whole ovarian lysates. ACTB (β actin) was used as a loading control. Data are representative of three independent experiments. (b) qRT-PCR analysis of VAMP7 mRNA levels normalized to GAPDH in human adult tissues. n = 6 independent samples for each tissue. Data are presented as means ± s.e.m. (ce) Immunohistological detection of VAMP7 in normal human adult testicular (c) and penile tissues including urethral (d) and preputial (e) epithelia. Scale bars, 125 μm. (f,g) Immunohistochemical detection of VAMP7 expression in fetal testis (f) and external genitalia (g) from WT male mice at embryonic day 16.5 (E16.5). Scale bars, 125 μm. The images are representative of three different male mouse embryos.

  3. Mice overexpressing VAMP7 exhibit cryptorchidism and abnormal external genitalia.
    Figure 3: Mice overexpressing VAMP7 exhibit cryptorchidism and abnormal external genitalia.

    (a) qRT-PCR analysis of VAMP7 mRNA levels normalized to Gapdh in testes from WT mice (n = 4 animals) and humanized VAMP7-BAC transgenic (V7BAC) mice (n = 5 mice for line 7 and n = 4 mice for line 21) using primers common to both mouse and human VAMP7 transcripts. Data are presented as means ± s.e.m. (b) VAMP7 immunohistological staining of fetal and adult testicular tissues and external genitalia from V7BAC and WT mice. Data are representative of four independent experiments. Scale bars, 125 μm. For fetal genital tubercles, scale bars, 500 μm. GT, genital tubercles. Adult ext. genit., adult external genitalia. (c) Distribution of unilateral and bilateral cryptorchidism in V7BAC mice harboring undescended testes. (n = 7 cryptorchid animals for each line). (d) Anatomical locations of cryptorchid testes in V7BAC mice (n = 14 cryptorchid testes for each line). Distribution is presented in percentage. (e) Representative pictures of testis position in V7BAC and WT mice. Images are representative of 14 independent mice. B, bladder; T, testis; Epid. fat, epidydimal fat. Scale bars, 1 cm. (f) Desmin staining of testis and gubernaculum of V7BAC and WT male embryos at E18.5. Images are representative of three independent experiments. Arrows indicate the gubernaculum cord. Scale bars, 500 μm. (g) H&E staining of cross-sections of genital tubercles from V7BAC male mouse embryos at E18.5 (n = 6) and WT littermates. Asterisks indicate normal and abnormal fusion of urethral folds or hypospadias, whereas arrows show abnormalities of the epithelial-lined prepuce housing the penis. The occurrence of penile defects is presented in percentage. Scale bar, 150 μm. (h) Measurement of anogenital distance in V7BAC adult male mice (n = 9) and WT littermates (n = 5). Data are presented as means ± s.e.m. (i) Measurement of penile length in V7BAC adult male mice (n = 5) compared to WT animals (n = 7). Data are presented as means ± s.e.m. Mean differences were determined by unpaired, two-tailed Student's t-test. ***P < 0.001.

  4. Elevated levels of VAMP7 modestly impair AR function.
    Figure 4: Elevated levels of VAMP7 modestly impair AR function.

    (a) Luciferase assays in HeLa cells cotransfected with a reporter vector driven by the androgen receptor response element of the kallikrein-related peptidase 3 promoter, and VAMP7 or VAMP7 and AR and incubated in the absence (ethanol vehicle) or in the presence of 1 × 10−8 M DHT for 24 h. n = 3 independent experiments for each condition. Data are presented as means ± s.e.m. One-way analysis of variance (ANOVA) with post hoc Bonferroni's test was used for statistical analyses. **P < 0.01. (b) Reciprocal coimmunoprecipitation of AR and VAMP7 following their cotransfection in HeLa cells. WB, western blot. Data are representative of three independent experiments. (c) Immunofluorescence staining of VAMP7, AR and RAB5 in HeLa cells after transfection with AR, VAMP7 or both, upon stimulation with ethanol (EtOH) or 1 × 10−8 M DHT for 24 h. Scale bars, 5 μm. Images are representative of three independent experiments. (d) AR immunostaining of adult testis from V7BAC or WT mice. Arrows indicate cytoplasmic staining. Scale bars, 125 μm (inset, 25 μm). Images are representative of three independent experiments. (e) In vivo chromatin immunoprecipitation assays of testicular lysates from WT (n = 3) and V7BAC (n = 3) mice using IgG or AR-specific antibody (Ab) followed by quantitative PCR of Fkbp5, Mafb and Fkbp4 promoters. Data are presented as means ± s.e.m. Two-way analysis of variance with post hoc Bonferroni's test was used for statistical analyses. **P < 0.01; ***P < 0.001. (f,g) Immunostaining of Fkbp5 (f) and Mafb (g) in fetal and adult external genitalia and testes of WT and VAMP7-BAC transgenic (V7BAC) mouse embryos (E18.5). For fetal tissues: scale bars, 250 μm. For adult urethra and testis: scale bars, 100 μm. Images are representative of three independent experiments.

  5. VAMP7 enhances estrogen receptor transcriptional activity.
    Figure 5: VAMP7 enhances estrogen receptor transcriptional activity.

    (a) Luciferase assays following transfection with a reporter construct containing estrogen receptor–responsive element and with VAMP7, ESR1 or VAMP7 plus ESR1 in HeLa cells incubated in absence (vehicle) or presence of 17β-estradiol (1 × 10−8 M) for 24 h. n = 3 independent experiments for each condition. RLU, relative light units. Data are presented as means ± s.e.m. One-way ANOVA with post hoc Bonferroni test was used for statistical analyses. ***P < 0.001. (b) Western blot analysis of ESR1 and lamin A/C (LMNA) in nuclear protein extracts of HeLa cells cotransfected with ESR1 or ESR1 plus VAMP7 (V7) in the absence (EtOH) or presence of 17β-estradiol (1 × 10−8 M) for 24 h. (c) Reciprocal coimmunoprecipitation of ESR1 and VAMP7 following their cotransfection in HeLa cells. IP, immunoprecipitation. (d) qRT-PCR analysis of key genes of ESR1 signaling in testes from WT (n = 3) and V7BAC mice (line 7; n = 3). v1 and v2, transcript variant 1 (NM_207707.1) and transcript variant 2 (NM_010157.3) of Esr2, respectively. Data are expressed as mean ± s.e.m. Mean differences between WT and V7BAC mice were determined by unpaired, two-tailed Student's t-test. *P < 0.05; **P < 0.01; ***P < 0.001. (e) ATF3 immunostaining of testis and external genitalia at fetal and adult stages of WT and V7BAC mice. For fetal tissues: scale bars, 250 μm. For adult urethra and testis: scale bars, 100 μm. Data are representative of three independent experiments. (f) qRT-PCR of VAMP7 and ATF3 gene expression after incubation with nontargeting (scramble) or VAMP7 siRNA in NT2/D1 cells. n = 3 independent experiments for each condition. Mean differences were determined by unpaired, two-tailed Student's t-test. *P < 0.05; **P < 0.01. (g) Left, heatmap of testis gene expression profiles in NT2/D1 cells transiently transfected with nontargeting (scramble) or VAMP7-specific siRNA. Yellow and blue colors indicate increased and decreased expression, respectively, relative to scramble. Right, Ingenuity Pathway Analysis (IPA 8.5) of top canonical pathways significantly altered (20% false discovery rate) after VAMP7 knockdown in NT2/D1 cells. Data are representative of three independent experiments. (h) Pictures of inguinal hernia in V7BAC males and H&E staining of extensive granulomatous inflammatory response of entrapped tissues. Scale bar, 100 μm. Data are representative of four independent experiments. (i) Schematic representation of VAMP7 impact on estrogen receptor signaling and male phenotypic development.

  6. Abnormal spermatogenesis and reduced motility and fertility in V7BAC mice.
    Figure 6: Abnormal spermatogenesis and reduced motility and fertility in V7BAC mice.

    (a) Weights of cryptorchid gonads (n = 7 testes) from 6-month-old male V7BAC mice and scrotal testes (n = 8 testes) from WT littermates. (b,c) Epididymis and seminal vesicles weights from 6-month-old male V7BAC (n = 8 mice) and WT (n = 6 mice) animals. (d) H&E-stained images of paraffin-embedded testis and caput and cauda epididymides sections from 6-month-old WT and V7BAC mice. Scale bars, 125 μm. Images are representative of three independent experiments. (eh) Serum testosterone (e), 17β-estradiol (f), Lh (g) and Fsh (h) hormone levels in 6-month-old male WT (n = 6 for each data point in each experiment) and V7BAC mice (n = 6 for each data point in each experiment). (i) Epidydimal sperm count and motility in 6-month-old WT (n = 6) and V7BAC (n = 5) mice. (j) Litter size from WT (n = 12 litters) and V7BAC (n = 16 litters) male mice in a 4-month continuous mating study. All data are expressed as mean ± s.e.m. Statistical significance was determined by unpaired, two-tailed Student's t-test. *P < 0.05; ***P < 0.001.

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

NCBI Reference Sequence

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Author information

  1. These authors contributed equally to this work.

    • Shuo Han &
    • Jean-Francois Louet

Affiliations

  1. Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA.

    • Mounia Tannour-Louet,
    • Karina Romero,
    • Josephine Addai,
    • Aysegul Sahin &
    • Dolores J Lamb
  2. Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, Sophia Antipolis, France.

    • Mounia Tannour-Louet
  3. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.

    • Shuo Han,
    • Jean-Francois Louet,
    • Bin Zhang &
    • Dolores J Lamb
  4. Centre Méditerranéen de Médecine Moléculaire, INSERM U1065, Nice, France.

    • Jean-Francois Louet
  5. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

    • Sau Wai Cheung
  6. Center for Reproductive Medicine, Baylor College of Medicine, Houston, Texas, USA.

    • Dolores J Lamb

Contributions

Both senior authors, M.T.-L. and D.J.L., conceived and supervised the study, conducted experiments, analyzed data and wrote and revised the manuscript. S.H. and J.-F.L. performed experiments and analyzed data. B.Z., K.R., J.A. and A.S. performed experiments. S.W.C. conducted the human CGH array studies.

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The authors declare no competing financial interests.

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