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

Tannour-Louet M, Han S, Louet JF, Zhang B, Romero K, Addai J, Sahin A, Cheung SW, Lamb DJ - Nat. Med. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA. [2] Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, Sophia Antipolis, France.

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.

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Mice overexpressing VAMP7 exhibit cryptorchidism and abnormal external genitalia. (a) qRT-PCR analysis of VAMP7 mRNA levels normalized to Gapdh in testis from WT (n = 4 animals) and humanized 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 tissue and external genitalia from V7BAC transgenic and WT mice. Scale bars, 125 µm. For fetal genital tubercles, scale bar, 500 µm. (c)Distribution of unilateral and bilateral cryptorchidism in V7BAC transgenic mice harboring undescended testes. (n = 7 cryptorchid animals for each line). (d) Anatomical locations of cryptorchid testes in VAMP7 transgenic mice (n = 14 cryptorchid testes for each line). Distribution is presented in percentage. (e) Representative pictures of testis position in V7BAC and WT mice. B: bladder; T: testis; Epid. fat: epidydimal fat. Scale bar, 1 cm. (f) Desmin staining of testis and gubernaculum of V7BAC and WT male embryos at gestational age E18.5. Arrows indicate the gubernaculum cord. Scale bar, 500 µm. (g) Hematoxylin-eosin staining of cross-sections of genital tubercles from V7BAC male mouse embryos at gestational age E18.5 (n = 6) and WT littermates. Asterisks indicate normal and abnormal fusion of urethral folds or hypospadias, while arrows show abnormalities of the epithelial-lined prepuce housing the penis. The occurrence of penile defects was presented in percentage. Scale bar, 150 µm. (h) Measurement of anogenital distance in VAMP7 transgenic adult male mice (n = 9) and WT littermates (n = 5). Data are presented as means ± s.e.m. (i) Measurement of penile length in VAMP7 mutant 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 < 0.05; **P < 0.01; ***P < 0.001.
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Figure 3: Mice overexpressing VAMP7 exhibit cryptorchidism and abnormal external genitalia. (a) qRT-PCR analysis of VAMP7 mRNA levels normalized to Gapdh in testis from WT (n = 4 animals) and humanized 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 tissue and external genitalia from V7BAC transgenic and WT mice. Scale bars, 125 µm. For fetal genital tubercles, scale bar, 500 µm. (c)Distribution of unilateral and bilateral cryptorchidism in V7BAC transgenic mice harboring undescended testes. (n = 7 cryptorchid animals for each line). (d) Anatomical locations of cryptorchid testes in VAMP7 transgenic mice (n = 14 cryptorchid testes for each line). Distribution is presented in percentage. (e) Representative pictures of testis position in V7BAC and WT mice. B: bladder; T: testis; Epid. fat: epidydimal fat. Scale bar, 1 cm. (f) Desmin staining of testis and gubernaculum of V7BAC and WT male embryos at gestational age E18.5. Arrows indicate the gubernaculum cord. Scale bar, 500 µm. (g) Hematoxylin-eosin staining of cross-sections of genital tubercles from V7BAC male mouse embryos at gestational age E18.5 (n = 6) and WT littermates. Asterisks indicate normal and abnormal fusion of urethral folds or hypospadias, while arrows show abnormalities of the epithelial-lined prepuce housing the penis. The occurrence of penile defects was presented in percentage. Scale bar, 150 µm. (h) Measurement of anogenital distance in VAMP7 transgenic adult male mice (n = 9) and WT littermates (n = 5). Data are presented as means ± s.e.m. (i) Measurement of penile length in VAMP7 mutant 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 < 0.05; **P < 0.01; ***P < 0.001.

Mentions: We sought to investigate in vivo the impact of genetically elevated VAMP7 levels on genitourinary development and reproductive physiology. A mouse model that expresses human VAMP7 under its endogenous regulatory sequences was produced to recapitulate the genetic gain observed in patients. Mutant mice were generated through microinjection of a 159-kilobase linearized bacterial artificial chromosome (BAC; clone RP11-479B17) including the human VAMP7 gene, into fertilized oocytes of FVB mice. Two independent VAMP7 founder lines, 7 and 21, were obtained. BAC transgenic mice were viable and had normal growth and lifespan. Western blot analysis using an antibody that specifically recognizes the human VAMP7 protein confirmed the expression of the transgene in the testes of mutant mice (Supplemental Fig. S3a). Using primers that recognize both human and mouse transcripts, we quantified mRNA levels of VAMP7 by qRT-PCR in the two lines (21 and 7) and found them to be only 2 to 3 times higher than the messenger levels of the endogenous Vamp7 in WT mice (Fig. 3a). Throughout the adult and fetal testes and external genitalia, the spatial expression pattern of the transgene mirrored that of the endogenous product in the wild-type animals and localized to cytoplasmic compartments as expected (Fig. 3b). A parallel study of the two transgenic lines controlled for potential insertion effects. Data from strain 7 are presented when identical outcomes were obtained for both lines (for line 21-see Supplemental Fig. S4).


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

Tannour-Louet M, Han S, Louet JF, Zhang B, Romero K, Addai J, Sahin A, Cheung SW, Lamb DJ - Nat. Med. (2014)

Mice overexpressing VAMP7 exhibit cryptorchidism and abnormal external genitalia. (a) qRT-PCR analysis of VAMP7 mRNA levels normalized to Gapdh in testis from WT (n = 4 animals) and humanized 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 tissue and external genitalia from V7BAC transgenic and WT mice. Scale bars, 125 µm. For fetal genital tubercles, scale bar, 500 µm. (c)Distribution of unilateral and bilateral cryptorchidism in V7BAC transgenic mice harboring undescended testes. (n = 7 cryptorchid animals for each line). (d) Anatomical locations of cryptorchid testes in VAMP7 transgenic mice (n = 14 cryptorchid testes for each line). Distribution is presented in percentage. (e) Representative pictures of testis position in V7BAC and WT mice. B: bladder; T: testis; Epid. fat: epidydimal fat. Scale bar, 1 cm. (f) Desmin staining of testis and gubernaculum of V7BAC and WT male embryos at gestational age E18.5. Arrows indicate the gubernaculum cord. Scale bar, 500 µm. (g) Hematoxylin-eosin staining of cross-sections of genital tubercles from V7BAC male mouse embryos at gestational age E18.5 (n = 6) and WT littermates. Asterisks indicate normal and abnormal fusion of urethral folds or hypospadias, while arrows show abnormalities of the epithelial-lined prepuce housing the penis. The occurrence of penile defects was presented in percentage. Scale bar, 150 µm. (h) Measurement of anogenital distance in VAMP7 transgenic adult male mice (n = 9) and WT littermates (n = 5). Data are presented as means ± s.e.m. (i) Measurement of penile length in VAMP7 mutant 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 < 0.05; **P < 0.01; ***P < 0.001.
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Figure 3: Mice overexpressing VAMP7 exhibit cryptorchidism and abnormal external genitalia. (a) qRT-PCR analysis of VAMP7 mRNA levels normalized to Gapdh in testis from WT (n = 4 animals) and humanized 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 tissue and external genitalia from V7BAC transgenic and WT mice. Scale bars, 125 µm. For fetal genital tubercles, scale bar, 500 µm. (c)Distribution of unilateral and bilateral cryptorchidism in V7BAC transgenic mice harboring undescended testes. (n = 7 cryptorchid animals for each line). (d) Anatomical locations of cryptorchid testes in VAMP7 transgenic mice (n = 14 cryptorchid testes for each line). Distribution is presented in percentage. (e) Representative pictures of testis position in V7BAC and WT mice. B: bladder; T: testis; Epid. fat: epidydimal fat. Scale bar, 1 cm. (f) Desmin staining of testis and gubernaculum of V7BAC and WT male embryos at gestational age E18.5. Arrows indicate the gubernaculum cord. Scale bar, 500 µm. (g) Hematoxylin-eosin staining of cross-sections of genital tubercles from V7BAC male mouse embryos at gestational age E18.5 (n = 6) and WT littermates. Asterisks indicate normal and abnormal fusion of urethral folds or hypospadias, while arrows show abnormalities of the epithelial-lined prepuce housing the penis. The occurrence of penile defects was presented in percentage. Scale bar, 150 µm. (h) Measurement of anogenital distance in VAMP7 transgenic adult male mice (n = 9) and WT littermates (n = 5). Data are presented as means ± s.e.m. (i) Measurement of penile length in VAMP7 mutant 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 < 0.05; **P < 0.01; ***P < 0.001.
Mentions: We sought to investigate in vivo the impact of genetically elevated VAMP7 levels on genitourinary development and reproductive physiology. A mouse model that expresses human VAMP7 under its endogenous regulatory sequences was produced to recapitulate the genetic gain observed in patients. Mutant mice were generated through microinjection of a 159-kilobase linearized bacterial artificial chromosome (BAC; clone RP11-479B17) including the human VAMP7 gene, into fertilized oocytes of FVB mice. Two independent VAMP7 founder lines, 7 and 21, were obtained. BAC transgenic mice were viable and had normal growth and lifespan. Western blot analysis using an antibody that specifically recognizes the human VAMP7 protein confirmed the expression of the transgene in the testes of mutant mice (Supplemental Fig. S3a). Using primers that recognize both human and mouse transcripts, we quantified mRNA levels of VAMP7 by qRT-PCR in the two lines (21 and 7) and found them to be only 2 to 3 times higher than the messenger levels of the endogenous Vamp7 in WT mice (Fig. 3a). Throughout the adult and fetal testes and external genitalia, the spatial expression pattern of the transgene mirrored that of the endogenous product in the wild-type animals and localized to cytoplasmic compartments as expected (Fig. 3b). A parallel study of the two transgenic lines controlled for potential insertion effects. Data from strain 7 are presented when identical outcomes were obtained for both lines (for line 21-see Supplemental Fig. S4).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA. [2] Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, Sophia Antipolis, France.

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.

Show MeSH
Related in: MedlinePlus