Figure 1.
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].
Figure 2.
Morphology of 2 month-old mice proximal epididymides.
(A) X-gal staining detecting endogenous β-galactosidase activity in a control mouse initial segment (IS) and proximal corpus (COR). Dicer1fl/fl; Defb41iCre/wt mice display no staining of initial segment and have enlarged efferent ducts (ED). (B) HE staining of the efferent ducts and the proximal epididymis of control and Dicer1fl/fl; Defb41iCre/wt mice. Scale bars 1.5 mm.
Table 1.
Fertility of Dicer1fl/fl; Defb41iCre/wt male mice.
Figure 3.
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.
Figure 4.
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.
Figure 5.
Cell proliferation and apoptosis.
Ki67 immunostaining; (A) Control; (B) Dicer1fl/fl; Defb41iCre/wt mouse. TUNEL labeling; (D) Control; (E) Dicer1fl/fl; Defb41iCre/wt mouse. Arrows mark apoptotic cells. (C, F) Comparison of number of proliferating cells and apoptotic cells in the initial segment (IS) and caput (CAP) of control (white bars) and Dicer1fl/fl; Defb41iCre/wt mice (grey bars). The number of proliferating and apoptotic cells were calculated from ten tubular cross sections of 5 control and 5 (Ki67 stained) and 8 (TUNEL labeled) Dicer1fl/fl; Defb41iCre/wt mice epididymides and the total cell number was divided by the circumference of the tubular cross sections (mm). Statistical significance of changes, calculated using the unpaired t-test, is indicated as follows: ns, not significant; *, P≤0.05; ***, P≤0.001. Scale bars 100 µm.
Expression of Sex steroid receptors.
(A) Immunostaining of two-month-old control mouse initial segment (IS) shows expression of Estrogen receptor 1 (ESR1) only in the narrow cells (indicated by arrows) while (B) the Dicer1fl/fl; Defb41iCre/wt mouse has ESR1 expression in most cells of the epithelium. (C, D) Staining for Androgen receptor. (D) Androgen receptor expression varied between Dicer1fl/fl; Defb41iCre/wt mice epididymides. Some samples had similar expression levels as the control while others showed areas of more weakly stained tissue. CAP, caput. Scale bars 100 µm. (E) Expression of androgen receptor (Ar) and estrogen receptor 1 and 2 (Esr1 and Esr2) as well as (F) AR target gene: glutathione peroxidase 5 (Gpx5), lipocalin 5 (Lcn5), cysteine-rich secretory protein 1 (Crisp1) mRNA in IS and CAP of 2 month-old control and Dicer1 conditional knock-out (cKO) mice epididymides. Expression values relative to L19 expression. 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: ns, not significant; *, P≤0.05; ***, P≤0.001.