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Dicer1 ablation in the mouse epididymis causes dedifferentiation of the epithelium and imbalance in sex steroid signaling.

Björkgren I, Saastamoinen L, Krutskikh A, Huhtaniemi I, Poutanen M, Sipilä P - PLoS ONE (2012)

Bottom Line: The postnatal development of the epididymis is a complex process that results in a highly differentiated epithelium, divided into several segments.The dedifferentiated epithelium also showed an increase in estrogen receptor 1 expression while the expression of androgen receptor and its target genes; glutathione peroxidase 5, lipocalin 5 and cysteine-rich secretory protein 1 was downregulated, indicating imbalanced sex steroid signaling.At the time of the final epididymal development, Dicer1 acts as a regulator of signaling pathways essential for maintaining epithelial cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute of Biomedicine, University of Turku, Turku, Finland.

ABSTRACT

Background: The postnatal development of the epididymis is a complex process that results in a highly differentiated epithelium, divided into several segments. Recent studies indicate a role for RNA interference (RNAi) in the development of the epididymis, however, the actual requirement for RNAi has remained elusive. Here, we present the first evidence of a direct need for RNAi in the differentiation of the epididymal epithelium.

Methodology/principal findings: By utilizing the Cre-LoxP system we have generated a conditional knock-out of Dicer1 in the two most proximal segments of the mouse epididymis. Recombination of Dicer1, catalyzed by Defb41(iCre/wt), took place before puberty, starting from 12 days postpartum. Shortly thereafter, downregulation of the expression of two genes specific for the most proximal epididymis (lipocalin 8 and cystatin 8) was observed. Following this, segment development continued until week 5 at which age the epithelium started to regress back to an undifferentiated state. The dedifferentiated epithelium also showed an increase in estrogen receptor 1 expression while the expression of androgen receptor and its target genes; glutathione peroxidase 5, lipocalin 5 and cysteine-rich secretory protein 1 was downregulated, indicating imbalanced sex steroid signaling.

Conclusions/significance: At the time of the final epididymal development, Dicer1 acts as a regulator of signaling pathways essential for maintaining epithelial cell differentiation.

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Related in: MedlinePlus

Immunohistochemical staining of the different epithelial cell types.Staining of initial segment (A, B) and initial segment and caput (C–H) of two-month-old control and Dicer1fl/fl; Defb41iCre/wt mouse epididymides. (A, B) Staining of principal cell F-actin by phalloidin-TRITC. (C, D) Expression of vacuolar H+-ATPase (V-ATPase) in narrow and clear cells (marked with arrows). (E, F) Expression of keratin 5 (Krt5) in basal cells. (G, H) Smooth muscle cells. Dicer1fl/fl; Defb41iCre/wt mice display a thicker smooth muscle cell layer than that of control mice. Inserts are 3× enlargements of the stained cells. (I) qRT-PCR for proximal epididymis-specific gene expression. Expression of lipocalin 8 (Lcn8), brain expressed myelocytomatosis oncogene (Bmyc), cystatin 8 (Cst8), Ros1 proto-oncogene (Ros1) and G protein-coupled receptor 64 (Gpr64) in the initial segment and caput of 2 month-old control and Dicer1 conditional knock-out (cKO) mouse epididymides. Values relative to L19 expression. Statistical significance was calculated from the expression levels of 3 control and 3 Dicer1fl/fl;Defb41iCre/wt mouse (Ros1∶8 control and 9 Dicer1fl/fl;Defb41iCre/wt mouse) samples using the unpaired t-test. Statistical significance of changes is indicated as follows: ns, not significant; *, P≤0.05; **, P≤0.01. Scale bars 100 µm.
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pone-0038457-g004: Immunohistochemical staining of the different epithelial cell types.Staining of initial segment (A, B) and initial segment and caput (C–H) of two-month-old control and Dicer1fl/fl; Defb41iCre/wt mouse epididymides. (A, B) Staining of principal cell F-actin by phalloidin-TRITC. (C, D) Expression of vacuolar H+-ATPase (V-ATPase) in narrow and clear cells (marked with arrows). (E, F) Expression of keratin 5 (Krt5) in basal cells. (G, H) Smooth muscle cells. Dicer1fl/fl; Defb41iCre/wt mice display a thicker smooth muscle cell layer than that of control mice. Inserts are 3× enlargements of the stained cells. (I) qRT-PCR for proximal epididymis-specific gene expression. Expression of lipocalin 8 (Lcn8), brain expressed myelocytomatosis oncogene (Bmyc), cystatin 8 (Cst8), Ros1 proto-oncogene (Ros1) and G protein-coupled receptor 64 (Gpr64) in the initial segment and caput of 2 month-old control and Dicer1 conditional knock-out (cKO) mouse epididymides. Values relative to L19 expression. Statistical significance was calculated from the expression levels of 3 control and 3 Dicer1fl/fl;Defb41iCre/wt mouse (Ros1∶8 control and 9 Dicer1fl/fl;Defb41iCre/wt mouse) samples using the unpaired t-test. Statistical significance of changes is indicated as follows: ns, not significant; *, P≤0.05; **, P≤0.01. Scale bars 100 µm.

Mentions: To study further the effect of Dicer1 ablation on epithelial cell differentiation, the presence of different epithelial cell types of the epididymal epithelium was analyzed by immunohistochemistry. Phalloidin conjugated to TRITC was used to stain the F-actin of principal cells, and antibodies against vacuolar H+-ATPase (V-ATPase) and Keratin 5 (Krt5) were used to stain clear/narrow cells and basal cells, respectively. The results indicated that all cell types were present in the Dicer1 cKO epididymis (Figure 4). The epithelial cell types were found in similar numbers and locations in the Dicer1 cKO epithelium as in control animals. However, α-actin staining revealed an increase in muscle cell layer thickness (Figure 4G, H). The Dicer1fl/fl; Defb41iCre/wt mice displayed three layers of muscle cells surrounding the epididymal duct, whereas control mice typically had one.


Dicer1 ablation in the mouse epididymis causes dedifferentiation of the epithelium and imbalance in sex steroid signaling.

Björkgren I, Saastamoinen L, Krutskikh A, Huhtaniemi I, Poutanen M, Sipilä P - PLoS ONE (2012)

Immunohistochemical staining of the different epithelial cell types.Staining of initial segment (A, B) and initial segment and caput (C–H) of two-month-old control and Dicer1fl/fl; Defb41iCre/wt mouse epididymides. (A, B) Staining of principal cell F-actin by phalloidin-TRITC. (C, D) Expression of vacuolar H+-ATPase (V-ATPase) in narrow and clear cells (marked with arrows). (E, F) Expression of keratin 5 (Krt5) in basal cells. (G, H) Smooth muscle cells. Dicer1fl/fl; Defb41iCre/wt mice display a thicker smooth muscle cell layer than that of control mice. Inserts are 3× enlargements of the stained cells. (I) qRT-PCR for proximal epididymis-specific gene expression. Expression of lipocalin 8 (Lcn8), brain expressed myelocytomatosis oncogene (Bmyc), cystatin 8 (Cst8), Ros1 proto-oncogene (Ros1) and G protein-coupled receptor 64 (Gpr64) in the initial segment and caput of 2 month-old control and Dicer1 conditional knock-out (cKO) mouse epididymides. Values relative to L19 expression. Statistical significance was calculated from the expression levels of 3 control and 3 Dicer1fl/fl;Defb41iCre/wt mouse (Ros1∶8 control and 9 Dicer1fl/fl;Defb41iCre/wt mouse) samples using the unpaired t-test. Statistical significance of changes is indicated as follows: ns, not significant; *, P≤0.05; **, P≤0.01. Scale bars 100 µm.
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Related In: Results  -  Collection

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pone-0038457-g004: Immunohistochemical staining of the different epithelial cell types.Staining of initial segment (A, B) and initial segment and caput (C–H) of two-month-old control and Dicer1fl/fl; Defb41iCre/wt mouse epididymides. (A, B) Staining of principal cell F-actin by phalloidin-TRITC. (C, D) Expression of vacuolar H+-ATPase (V-ATPase) in narrow and clear cells (marked with arrows). (E, F) Expression of keratin 5 (Krt5) in basal cells. (G, H) Smooth muscle cells. Dicer1fl/fl; Defb41iCre/wt mice display a thicker smooth muscle cell layer than that of control mice. Inserts are 3× enlargements of the stained cells. (I) qRT-PCR for proximal epididymis-specific gene expression. Expression of lipocalin 8 (Lcn8), brain expressed myelocytomatosis oncogene (Bmyc), cystatin 8 (Cst8), Ros1 proto-oncogene (Ros1) and G protein-coupled receptor 64 (Gpr64) in the initial segment and caput of 2 month-old control and Dicer1 conditional knock-out (cKO) mouse epididymides. Values relative to L19 expression. Statistical significance was calculated from the expression levels of 3 control and 3 Dicer1fl/fl;Defb41iCre/wt mouse (Ros1∶8 control and 9 Dicer1fl/fl;Defb41iCre/wt mouse) samples using the unpaired t-test. Statistical significance of changes is indicated as follows: ns, not significant; *, P≤0.05; **, P≤0.01. Scale bars 100 µm.
Mentions: To study further the effect of Dicer1 ablation on epithelial cell differentiation, the presence of different epithelial cell types of the epididymal epithelium was analyzed by immunohistochemistry. Phalloidin conjugated to TRITC was used to stain the F-actin of principal cells, and antibodies against vacuolar H+-ATPase (V-ATPase) and Keratin 5 (Krt5) were used to stain clear/narrow cells and basal cells, respectively. The results indicated that all cell types were present in the Dicer1 cKO epididymis (Figure 4). The epithelial cell types were found in similar numbers and locations in the Dicer1 cKO epithelium as in control animals. However, α-actin staining revealed an increase in muscle cell layer thickness (Figure 4G, H). The Dicer1fl/fl; Defb41iCre/wt mice displayed three layers of muscle cells surrounding the epididymal duct, whereas control mice typically had one.

Bottom Line: The postnatal development of the epididymis is a complex process that results in a highly differentiated epithelium, divided into several segments.The dedifferentiated epithelium also showed an increase in estrogen receptor 1 expression while the expression of androgen receptor and its target genes; glutathione peroxidase 5, lipocalin 5 and cysteine-rich secretory protein 1 was downregulated, indicating imbalanced sex steroid signaling.At the time of the final epididymal development, Dicer1 acts as a regulator of signaling pathways essential for maintaining epithelial cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Institute of Biomedicine, University of Turku, Turku, Finland.

ABSTRACT

Background: The postnatal development of the epididymis is a complex process that results in a highly differentiated epithelium, divided into several segments. Recent studies indicate a role for RNA interference (RNAi) in the development of the epididymis, however, the actual requirement for RNAi has remained elusive. Here, we present the first evidence of a direct need for RNAi in the differentiation of the epididymal epithelium.

Methodology/principal findings: By utilizing the Cre-LoxP system we have generated a conditional knock-out of Dicer1 in the two most proximal segments of the mouse epididymis. Recombination of Dicer1, catalyzed by Defb41(iCre/wt), took place before puberty, starting from 12 days postpartum. Shortly thereafter, downregulation of the expression of two genes specific for the most proximal epididymis (lipocalin 8 and cystatin 8) was observed. Following this, segment development continued until week 5 at which age the epithelium started to regress back to an undifferentiated state. The dedifferentiated epithelium also showed an increase in estrogen receptor 1 expression while the expression of androgen receptor and its target genes; glutathione peroxidase 5, lipocalin 5 and cysteine-rich secretory protein 1 was downregulated, indicating imbalanced sex steroid signaling.

Conclusions/significance: At the time of the final epididymal development, Dicer1 acts as a regulator of signaling pathways essential for maintaining epithelial cell differentiation.

Show MeSH
Related in: MedlinePlus