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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

The epididymis specific Dicer1 knock-out.(A) Schematic diagram of the Dicer1 locus with loxP sites flanking exon 24 (e24). Arrows indicate the location of genotyping primers used for analyzing the deletion of e24. (B) Genomic PCR of 12 day-old mice showing the intact locus (Dicer1fl) and the recombinant locus with e24 deleted (Dicer1flΔ). Exon 24 is only deleted in the Dicer1fl/fl; Defb41iCre/wt mouse initial segment (IS) and caput (CAP) while the iCre locus is detected in all segments of the epididymis. (C) Expression of Dicer1 mRNA in the whole epididymis of 1–42 day-old wild-type mice. (D) Dicer1 mRNA expression levels in the efferent ducts (ED), the different segments of the epididymis and testis (TE) of 2 month-old control and Dicer1 conditional knock-out (cKO) mice. Expression levels are presented relative to Ppia (testis) and L19 (epididymis) expression. COR, corpus; CAU, cauda. Statistical significance was calculated from the expression levels of 3 control and 4 Dicer1fl/fl; Defb41iCre/wt mouse samples using the unpaired t-test. Statistical significance of changes is indicated as follows: **, P≤0.01. Schematic picture modified from Harfe et al., 2005 [60].
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pone-0038457-g001: The epididymis specific Dicer1 knock-out.(A) Schematic diagram of the Dicer1 locus with loxP sites flanking exon 24 (e24). Arrows indicate the location of genotyping primers used for analyzing the deletion of e24. (B) Genomic PCR of 12 day-old mice showing the intact locus (Dicer1fl) and the recombinant locus with e24 deleted (Dicer1flΔ). Exon 24 is only deleted in the Dicer1fl/fl; Defb41iCre/wt mouse initial segment (IS) and caput (CAP) while the iCre locus is detected in all segments of the epididymis. (C) Expression of Dicer1 mRNA in the whole epididymis of 1–42 day-old wild-type mice. (D) Dicer1 mRNA expression levels in the efferent ducts (ED), the different segments of the epididymis and testis (TE) of 2 month-old control and Dicer1 conditional knock-out (cKO) mice. Expression levels are presented relative to Ppia (testis) and L19 (epididymis) expression. COR, corpus; CAU, cauda. Statistical significance was calculated from the expression levels of 3 control and 4 Dicer1fl/fl; Defb41iCre/wt mouse samples using the unpaired t-test. Statistical significance of changes is indicated as follows: **, P≤0.01. Schematic picture modified from Harfe et al., 2005 [60].

Mentions: Quantitative RT-PCR of the mouse epididymis showed a continuous expression of Dicer1 from birth into adulthood (Figure 1C). As the full knock-out of Dicer1 is embryonically lethal [21], we generated a Dicer1 cKO mouse line by crossing Dicer1fl/fl mice [29] with a mouse line expressing iCre under the Defensin beta 41 (Defb41) promoter. The Dicer1fl allele consists of two loxP sites flanking exon 24, which contains a major part of the second RNaseIII domain (Figure 1A). The heterozygous Defb41iCre mouse line did not show any phenotypic defects or fertility problems and expressed iCre in the epithelium of the most proximal part of the epididymis, IS and CAP. Recombination of Dicer1 was observed in 12 day-old Dicer1fl/fl; Defb41iCre/wt mouse IS and CAP by genomic PCR (Figure 1B) and qRT-PCR studies revealed a significant reduction in Dicer1 expression levels at the age of 2 months (Figure 1D).


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)

The epididymis specific Dicer1 knock-out.(A) Schematic diagram of the Dicer1 locus with loxP sites flanking exon 24 (e24). Arrows indicate the location of genotyping primers used for analyzing the deletion of e24. (B) Genomic PCR of 12 day-old mice showing the intact locus (Dicer1fl) and the recombinant locus with e24 deleted (Dicer1flΔ). Exon 24 is only deleted in the Dicer1fl/fl; Defb41iCre/wt mouse initial segment (IS) and caput (CAP) while the iCre locus is detected in all segments of the epididymis. (C) Expression of Dicer1 mRNA in the whole epididymis of 1–42 day-old wild-type mice. (D) Dicer1 mRNA expression levels in the efferent ducts (ED), the different segments of the epididymis and testis (TE) of 2 month-old control and Dicer1 conditional knock-out (cKO) mice. Expression levels are presented relative to Ppia (testis) and L19 (epididymis) expression. COR, corpus; CAU, cauda. Statistical significance was calculated from the expression levels of 3 control and 4 Dicer1fl/fl; Defb41iCre/wt mouse samples using the unpaired t-test. Statistical significance of changes is indicated as follows: **, P≤0.01. Schematic picture modified from Harfe et al., 2005 [60].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368854&req=5

pone-0038457-g001: The epididymis specific Dicer1 knock-out.(A) Schematic diagram of the Dicer1 locus with loxP sites flanking exon 24 (e24). Arrows indicate the location of genotyping primers used for analyzing the deletion of e24. (B) Genomic PCR of 12 day-old mice showing the intact locus (Dicer1fl) and the recombinant locus with e24 deleted (Dicer1flΔ). Exon 24 is only deleted in the Dicer1fl/fl; Defb41iCre/wt mouse initial segment (IS) and caput (CAP) while the iCre locus is detected in all segments of the epididymis. (C) Expression of Dicer1 mRNA in the whole epididymis of 1–42 day-old wild-type mice. (D) Dicer1 mRNA expression levels in the efferent ducts (ED), the different segments of the epididymis and testis (TE) of 2 month-old control and Dicer1 conditional knock-out (cKO) mice. Expression levels are presented relative to Ppia (testis) and L19 (epididymis) expression. COR, corpus; CAU, cauda. Statistical significance was calculated from the expression levels of 3 control and 4 Dicer1fl/fl; Defb41iCre/wt mouse samples using the unpaired t-test. Statistical significance of changes is indicated as follows: **, P≤0.01. Schematic picture modified from Harfe et al., 2005 [60].
Mentions: Quantitative RT-PCR of the mouse epididymis showed a continuous expression of Dicer1 from birth into adulthood (Figure 1C). As the full knock-out of Dicer1 is embryonically lethal [21], we generated a Dicer1 cKO mouse line by crossing Dicer1fl/fl mice [29] with a mouse line expressing iCre under the Defensin beta 41 (Defb41) promoter. The Dicer1fl allele consists of two loxP sites flanking exon 24, which contains a major part of the second RNaseIII domain (Figure 1A). The heterozygous Defb41iCre mouse line did not show any phenotypic defects or fertility problems and expressed iCre in the epithelium of the most proximal part of the epididymis, IS and CAP. Recombination of Dicer1 was observed in 12 day-old Dicer1fl/fl; Defb41iCre/wt mouse IS and CAP by genomic PCR (Figure 1B) and qRT-PCR studies revealed a significant reduction in Dicer1 expression levels at the age of 2 months (Figure 1D).

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