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Targeted inactivation of the androgen receptor gene in murine proximal epididymis causes epithelial hypotrophy and obstructive azoospermia.

Krutskikh A, De Gendt K, Sharp V, Verhoeven G, Poutanen M, Huhtaniemi I - Endocrinology (2010)

Bottom Line: The epithelial lining of the epididymal duct expresses the androgen receptor (Ar) along its entire length and undergoes rapid and profound degeneration when androgenic support is withdrawn.At 20-25 d of life, on the onset of Rnase10 expression, Ar became selectively inactivated in the principal cells of proximal epididymis, resulting in epithelial hypoplasia and hypotrophy.Our findings demonstrate that the development and function of the epididymal initial segment is critically dependent on direct androgen regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, London W12 0NN, United Kingdom.

ABSTRACT
The epithelial lining of the epididymal duct expresses the androgen receptor (Ar) along its entire length and undergoes rapid and profound degeneration when androgenic support is withdrawn. However, experiments involving orchidectomy with systemic testosterone replacement, and testicular efferent duct ligation, have indicated that structural and functional integrity of the initial segment cannot be maintained by circulating androgen alone, leaving the role of androgen in this epididymal zone unclear. We addressed this question in a mouse model with intact testicular output and selective Ar inactivation in the proximal epididymis by creating double-transgenic males carrying a conditional Ar(loxP) allele and expressing Cre recombinase under the promoter of Rnase10, a gene specifically expressed in proximal epididymis. At 20-25 d of life, on the onset of Rnase10 expression, Ar became selectively inactivated in the principal cells of proximal epididymis, resulting in epithelial hypoplasia and hypotrophy. Upon the subsequent onset of spermiation, epididymal obstruction ensued, with the consequent development of spermatic granulomata, back pressure-induced atrophy of the seminiferous epithelium, orchitis, and fibrosis of the testicular parenchyma. Consistent with these findings, the mice were infertile. When the effect of Ar knockout on gene expression in the proximal epididymis was compared with that of efferent duct ligation and orchidectomy, we identified genes specifically regulated by androgen, testicular efferent fluid, and both. Our findings demonstrate that the development and function of the epididymal initial segment is critically dependent on direct androgen regulation. The phenotype of the produced knockout mouse provides a novel model for obstructive azoospermia.

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Histological appearance and immunohistochemical detection of AR protein in reproductive organs of ProxE-ARKO mice. At 40 dpp: A, testis (S, Sertoli cells; L, Leydig cells); B, efferent ducts; C–I, epididymis segments IV-X; at 60 dpp: J, vas deferens; K, seminal vesicle; L, coagulating gland; M, ampullary gland; N, ventral prostate; O, dorsal prostate. Bar, 50 μm (counterstaining with hematoxylin). Immunodetection of AR in WT male reproductive tract is shown in Supplemental Fig. 4.
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Figure 3: Histological appearance and immunohistochemical detection of AR protein in reproductive organs of ProxE-ARKO mice. At 40 dpp: A, testis (S, Sertoli cells; L, Leydig cells); B, efferent ducts; C–I, epididymis segments IV-X; at 60 dpp: J, vas deferens; K, seminal vesicle; L, coagulating gland; M, ampullary gland; N, ventral prostate; O, dorsal prostate. Bar, 50 μm (counterstaining with hematoxylin). Immunodetection of AR in WT male reproductive tract is shown in Supplemental Fig. 4.

Mentions: The expression of Rnase10 starts at around 17 d postpartum (dpp) (6) coincidentally with the differentiation of IS. Although Rnase10 has previously been described to be expressed exclusively in the epididymal IS (6), the zone of Ar inactivation in the ProxE-ARKO mice appeared also to include the whole of segment II, continuing into segment III (Fig. 2B and Supplemental Fig. 1). The spatial pattern of Ar inactivation in the ProxE-ARKO epididymis was found to correspond to the area of endogenous Rnase10 expression as confirmed by RT-PCR (Fig. 1C). The zone of Ar inactivation began abruptly in the margin between the efferent ducts and IS and waned gradually distally, without a distinct border (Supplemental Fig. 1). Immunohistochemical analysis of proximal epididymides from prepubertal mice showed that in the ProxE-ARKO males AR began to disappear from the nuclei of epithelial cells between d 20 and 25 of life (Supplemental Fig. 2). We found that during this time window, proximal epithelium began to differentiate from simple cuboidal into columnar pseudostratified. The pseudostratification is a result of appearance of different cell types within the epithelium, and we observed ablation of AR in nuclei of the middle layer, i.e. in differentiating principal cells that started to express Rnase10 (Supplemental Fig. 2). Consequent to the disappearance of the AR, there was a loss of epithelial height, whereas further development of the IS was aborted. Accordingly, the principal cell-specific glutamate transporter excitatory amino acid carrier 1 (solute carrier family 1) (8), localized in stereocilia in IS and in microvilli in the more distal parts of the epididymis, was absent in the knockout IS but present in corpus and cauda (Supplemental Fig. 3). Ar expression remained intact in the rest of the Wolffian duct-derived structures, as well as in the testes and accessory sex glands (Fig. 3), and similar to that detected in WT control mice (Supplemental Fig. 4).


Targeted inactivation of the androgen receptor gene in murine proximal epididymis causes epithelial hypotrophy and obstructive azoospermia.

Krutskikh A, De Gendt K, Sharp V, Verhoeven G, Poutanen M, Huhtaniemi I - Endocrinology (2010)

Histological appearance and immunohistochemical detection of AR protein in reproductive organs of ProxE-ARKO mice. At 40 dpp: A, testis (S, Sertoli cells; L, Leydig cells); B, efferent ducts; C–I, epididymis segments IV-X; at 60 dpp: J, vas deferens; K, seminal vesicle; L, coagulating gland; M, ampullary gland; N, ventral prostate; O, dorsal prostate. Bar, 50 μm (counterstaining with hematoxylin). Immunodetection of AR in WT male reproductive tract is shown in Supplemental Fig. 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101806&req=5

Figure 3: Histological appearance and immunohistochemical detection of AR protein in reproductive organs of ProxE-ARKO mice. At 40 dpp: A, testis (S, Sertoli cells; L, Leydig cells); B, efferent ducts; C–I, epididymis segments IV-X; at 60 dpp: J, vas deferens; K, seminal vesicle; L, coagulating gland; M, ampullary gland; N, ventral prostate; O, dorsal prostate. Bar, 50 μm (counterstaining with hematoxylin). Immunodetection of AR in WT male reproductive tract is shown in Supplemental Fig. 4.
Mentions: The expression of Rnase10 starts at around 17 d postpartum (dpp) (6) coincidentally with the differentiation of IS. Although Rnase10 has previously been described to be expressed exclusively in the epididymal IS (6), the zone of Ar inactivation in the ProxE-ARKO mice appeared also to include the whole of segment II, continuing into segment III (Fig. 2B and Supplemental Fig. 1). The spatial pattern of Ar inactivation in the ProxE-ARKO epididymis was found to correspond to the area of endogenous Rnase10 expression as confirmed by RT-PCR (Fig. 1C). The zone of Ar inactivation began abruptly in the margin between the efferent ducts and IS and waned gradually distally, without a distinct border (Supplemental Fig. 1). Immunohistochemical analysis of proximal epididymides from prepubertal mice showed that in the ProxE-ARKO males AR began to disappear from the nuclei of epithelial cells between d 20 and 25 of life (Supplemental Fig. 2). We found that during this time window, proximal epithelium began to differentiate from simple cuboidal into columnar pseudostratified. The pseudostratification is a result of appearance of different cell types within the epithelium, and we observed ablation of AR in nuclei of the middle layer, i.e. in differentiating principal cells that started to express Rnase10 (Supplemental Fig. 2). Consequent to the disappearance of the AR, there was a loss of epithelial height, whereas further development of the IS was aborted. Accordingly, the principal cell-specific glutamate transporter excitatory amino acid carrier 1 (solute carrier family 1) (8), localized in stereocilia in IS and in microvilli in the more distal parts of the epididymis, was absent in the knockout IS but present in corpus and cauda (Supplemental Fig. 3). Ar expression remained intact in the rest of the Wolffian duct-derived structures, as well as in the testes and accessory sex glands (Fig. 3), and similar to that detected in WT control mice (Supplemental Fig. 4).

Bottom Line: The epithelial lining of the epididymal duct expresses the androgen receptor (Ar) along its entire length and undergoes rapid and profound degeneration when androgenic support is withdrawn.At 20-25 d of life, on the onset of Rnase10 expression, Ar became selectively inactivated in the principal cells of proximal epididymis, resulting in epithelial hypoplasia and hypotrophy.Our findings demonstrate that the development and function of the epididymal initial segment is critically dependent on direct androgen regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, London W12 0NN, United Kingdom.

ABSTRACT
The epithelial lining of the epididymal duct expresses the androgen receptor (Ar) along its entire length and undergoes rapid and profound degeneration when androgenic support is withdrawn. However, experiments involving orchidectomy with systemic testosterone replacement, and testicular efferent duct ligation, have indicated that structural and functional integrity of the initial segment cannot be maintained by circulating androgen alone, leaving the role of androgen in this epididymal zone unclear. We addressed this question in a mouse model with intact testicular output and selective Ar inactivation in the proximal epididymis by creating double-transgenic males carrying a conditional Ar(loxP) allele and expressing Cre recombinase under the promoter of Rnase10, a gene specifically expressed in proximal epididymis. At 20-25 d of life, on the onset of Rnase10 expression, Ar became selectively inactivated in the principal cells of proximal epididymis, resulting in epithelial hypoplasia and hypotrophy. Upon the subsequent onset of spermiation, epididymal obstruction ensued, with the consequent development of spermatic granulomata, back pressure-induced atrophy of the seminiferous epithelium, orchitis, and fibrosis of the testicular parenchyma. Consistent with these findings, the mice were infertile. When the effect of Ar knockout on gene expression in the proximal epididymis was compared with that of efferent duct ligation and orchidectomy, we identified genes specifically regulated by androgen, testicular efferent fluid, and both. Our findings demonstrate that the development and function of the epididymal initial segment is critically dependent on direct androgen regulation. The phenotype of the produced knockout mouse provides a novel model for obstructive azoospermia.

Show MeSH
Related in: MedlinePlus