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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.

Show MeSH
Differentiation of the epididymal epithelium.Hematoxylin and eosin staining of control and Dicer1fl/fl; Defb41iCre/wt mouse epididymides (A, B) The undifferentiated epithelium of the proximal epididymis of a 14 day-old control and a Dicer1fl/fl; Defb41iCre/wt mouse. (C, D) 33 day-old control and Dicer1fl/fl; Defb41iCre/wt mouse showing initiated differentiation of the initial segment (IS). (E) The fully developed IS of a 45 day-old control mouse. (F) The epithelium of a Dicer1fl/fl; Defb41iCre/wt mouse IS resembling that of the 14 day-old mouse. (G, H) The epididymis of an adult, 2 month-old, control and Dicer1fl/fl; Defb41iCre/wt mouse. CAP, caput. Scale bars 100 µm.
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pone-0038457-g003: Differentiation of the epididymal epithelium.Hematoxylin and eosin staining of control and Dicer1fl/fl; Defb41iCre/wt mouse epididymides (A, B) The undifferentiated epithelium of the proximal epididymis of a 14 day-old control and a Dicer1fl/fl; Defb41iCre/wt mouse. (C, D) 33 day-old control and Dicer1fl/fl; Defb41iCre/wt mouse showing initiated differentiation of the initial segment (IS). (E) The fully developed IS of a 45 day-old control mouse. (F) The epithelium of a Dicer1fl/fl; Defb41iCre/wt mouse IS resembling that of the 14 day-old mouse. (G, H) The epididymis of an adult, 2 month-old, control and Dicer1fl/fl; Defb41iCre/wt mouse. CAP, caput. Scale bars 100 µm.

Mentions: Macroscopic evaluation of 2 month-old Dicer1fl/fl; Defb41iCre/wt mice epididymides revealed an underdeveloped IS and, in addition, the mice frequently presented with enlarged efferent ducts (Figure 2A, B). The IS of control mice can be clearly visualized owing to the endogenous β-galactosidase activity in the segment. The much smaller IS of Dicer1fl/fl; Defb41iCre/wt mice could not be distinguished from CAP with X-gal staining (Figure 2A). Furthermore, the intense vasculature typical of WT IS was missing from the Dicer1 cKO IS. Histological evaluation showed a division of the epididymis into different segments (Figure 2B) but the epithelial cell layer of both IS and CAP was disorganized (Figure 3H). Dicer1fl/fl mice have a similar phenotype to WT mice epididymides and were used as controls throughout the study. Dicer1 cKO epididymides were significantly smaller than those of control mice (30.4±1.5 mg, control: 35.4±0.7 mg, P≤0.01). No significant difference in the weight of 6 month-old Dicer1 cKO and control mice epididymides was observed. However, the epithelial cell layer of the 6 month-old Dicer1fl/fl; Defb41iCre/wt mouse was further disturbed, with neoplastic changes in the efferent ducts causing their progressive obstruction (Figure S1). Even though sperm were detected in the CAU, 2- to 3-month-old Dicer1fl/fl; Defb41iCre/wt male mice failed to produce offspring when mated with WT females (Table 1). The number of sperm was reduced in Dicer1fl/fl; Defb41iCre/wt mouse epididymides as histological staining showed some tubular cross sections with no sperm. At 6 months of age the number of tubular cross sections without sperm was further increased due to the obstruction of the Dicer1fl/fl; Defb41iCre/wt mouse efferent ducts. The testis of the 6 month-old Dicer1fl/fl; Defb41iCre/wt mouse also displayed disruption of the seminiferous epithelium owing to fluid back-pressure (data not shown). Further morphological analyses revealed that the number of sperm with angulated tails was not significantly increased in 2 month-old Dicer1fl/fl; Defb41iCre/wt mouse CAU (22.9±3.4% of all sperm, control: 16.3±1.2%).


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)

Differentiation of the epididymal epithelium.Hematoxylin and eosin staining of control and Dicer1fl/fl; Defb41iCre/wt mouse epididymides (A, B) The undifferentiated epithelium of the proximal epididymis of a 14 day-old control and a Dicer1fl/fl; Defb41iCre/wt mouse. (C, D) 33 day-old control and Dicer1fl/fl; Defb41iCre/wt mouse showing initiated differentiation of the initial segment (IS). (E) The fully developed IS of a 45 day-old control mouse. (F) The epithelium of a Dicer1fl/fl; Defb41iCre/wt mouse IS resembling that of the 14 day-old mouse. (G, H) The epididymis of an adult, 2 month-old, control and Dicer1fl/fl; Defb41iCre/wt mouse. CAP, caput. Scale bars 100 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368854&req=5

pone-0038457-g003: Differentiation of the epididymal epithelium.Hematoxylin and eosin staining of control and Dicer1fl/fl; Defb41iCre/wt mouse epididymides (A, B) The undifferentiated epithelium of the proximal epididymis of a 14 day-old control and a Dicer1fl/fl; Defb41iCre/wt mouse. (C, D) 33 day-old control and Dicer1fl/fl; Defb41iCre/wt mouse showing initiated differentiation of the initial segment (IS). (E) The fully developed IS of a 45 day-old control mouse. (F) The epithelium of a Dicer1fl/fl; Defb41iCre/wt mouse IS resembling that of the 14 day-old mouse. (G, H) The epididymis of an adult, 2 month-old, control and Dicer1fl/fl; Defb41iCre/wt mouse. CAP, caput. Scale bars 100 µm.
Mentions: Macroscopic evaluation of 2 month-old Dicer1fl/fl; Defb41iCre/wt mice epididymides revealed an underdeveloped IS and, in addition, the mice frequently presented with enlarged efferent ducts (Figure 2A, B). The IS of control mice can be clearly visualized owing to the endogenous β-galactosidase activity in the segment. The much smaller IS of Dicer1fl/fl; Defb41iCre/wt mice could not be distinguished from CAP with X-gal staining (Figure 2A). Furthermore, the intense vasculature typical of WT IS was missing from the Dicer1 cKO IS. Histological evaluation showed a division of the epididymis into different segments (Figure 2B) but the epithelial cell layer of both IS and CAP was disorganized (Figure 3H). Dicer1fl/fl mice have a similar phenotype to WT mice epididymides and were used as controls throughout the study. Dicer1 cKO epididymides were significantly smaller than those of control mice (30.4±1.5 mg, control: 35.4±0.7 mg, P≤0.01). No significant difference in the weight of 6 month-old Dicer1 cKO and control mice epididymides was observed. However, the epithelial cell layer of the 6 month-old Dicer1fl/fl; Defb41iCre/wt mouse was further disturbed, with neoplastic changes in the efferent ducts causing their progressive obstruction (Figure S1). Even though sperm were detected in the CAU, 2- to 3-month-old Dicer1fl/fl; Defb41iCre/wt male mice failed to produce offspring when mated with WT females (Table 1). The number of sperm was reduced in Dicer1fl/fl; Defb41iCre/wt mouse epididymides as histological staining showed some tubular cross sections with no sperm. At 6 months of age the number of tubular cross sections without sperm was further increased due to the obstruction of the Dicer1fl/fl; Defb41iCre/wt mouse efferent ducts. The testis of the 6 month-old Dicer1fl/fl; Defb41iCre/wt mouse also displayed disruption of the seminiferous epithelium owing to fluid back-pressure (data not shown). Further morphological analyses revealed that the number of sperm with angulated tails was not significantly increased in 2 month-old Dicer1fl/fl; Defb41iCre/wt mouse CAU (22.9±3.4% of all sperm, control: 16.3±1.2%).

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