Epididymal Okajimas Folia basalAnat. cellsJpn., during mouse 83–89, development February, 2015 83 91(4):
(Original)
Cytokeratin localization and basal cell differentiation in the epididymal epithelium during postnatal development of the mouse By
Ayaka YOSHIDA, Kana MURAKAMI, Kentaro SAKUDA, and Kazuya YOSHINAGA Department of Anatomy and Cell Biology, Graduate School of Health Sciences, Kumamoto University, 4-24-1 Kuhonji, Kumamoto 862-0975, Japan – Received for Publication, January 19, 2015 – Key Words:
basal cells, cytokeratins, differentiation, immunohistochemistry, mouse epididymis
Summary: The epididymis is a male genital organ that has plays various functions, including sperm concentration, maturation, and storage. The epididymal epithelium consists of principal cells, clear cells, and basal cells. To comprehensively understand the occurrence and morphological differentiation of basal cells, we examined the expression and localization of cytokeratins (CKs) in the epididymal epithelium during postnatal development of the mouse. Immunohistochemical staining showed that, in adult mice, CK5 and CK14 were exclusively expressed in the cytoplasm of basal cells. During postnatal development, basal cells that stained positive for CK5 and CK14 first appeared in immature columnar epithelial cells in mice aged 1 week. The immunoreactivity became progressively stronger in mice aged 2–3 weeks. In mice aged 3 weeks, the immunoreactivity was strong in regions IV and V. In mice aged ≥ 4 weeks, strong immunoreactivity was observed in all epididymal regions. CK5 and CK14 could be useful markers of differentiation in epididymal basal cells. These basal cells originate from immature columnar epithelial cells and are of two types—dome-shaped and flask-shaped—. The flask-shaped cells are mainly located in the initial segment of the mouse epididymis.
Introduction The epididymis is a male genital organ that has a variety of functions, including concentration, maturation, protection, and storage of sperm1). Spermatozoa are formed in the testes and become functionally mature during their passage through the epididymal ducts, acquiring motility and a fertilizing capacity via biochemical and physiological changes1–3). The mouse epididymal duct can be divided into five regions—I (initial segment), II, III, IV, and V, according to the morphological properties of the epithelial cells in each region4) (Fig. 1). The epididymal epithelium consists mainly of principal cells, clear cells, and basal cells5–7). Functionally, it is known that principal cells contribute to absorption of luminal fluid and secretion of glycoproteins, and clear cells contribute to acidification of the lumen by secreting protons7–10). Although basal cells have been proposed to have potential roles as stem cells and sensor cells for monitoring the luminal environment11–13), their actual functions have not yet been determined.
Cytokeratins (CKs) comprise a large family of cytoskeletal intermediate-filament proteins, and the composition of CKs, with different structural components, varies according to the type or developmental stage of epithelial cells. Thus, CKs are, clinically, used as markers to diagnose epithelial tumors and to monitor the response to treatments. CKs are known to be expressed and localized in epididymal epithelial cells of mice14,15); however, little is known about the relationship between cytokeratin expression and the histological appearance and differentiation stage of basal cells. The present study was performed to elucidate the occurrence and localization of CKs in epididymal epithelium during postnatal development of the mouse, and to complement the few previous reports of the morphological differentiation of murine epididymal basal cells5, 14–16).
Corresponding author: Kazuya Yoshinaga, Department of Anatomy and Cell Biology, Graduate School of Health Sciences, Kumamoto University, 4-24-1 Kuhonji, Kumamoto 862-0975, Japan. E-mail: [email protected]
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ylin, progressively dehydrated, cleared, and mounted. Negative controls were obtained by omission of the primary antibodies.
Fig. 1.
Illustration of mouse epididymal regions. The adult mouse epididymis can be morphologically divided into five regions (I– V).
Materials and Methods Animals and tissue preparations Male C57BL/6 mice, differing in age by 1-week intervals from birth (week 0) to 13 weeks of age, were purchased from Japan SLC (Hamamatsu, Japan). The experiments using laboratory mice were approved by the Committee on Animal Research at Kumamoto University. At least three animals of each age were used. Each epididymis was fixed overnight at 4°C by perfusion with, or immersion in, 4% paraformaldehyde, embedded in paraffin, and 3- to 4-μm-thick sections were prepared. Immunohistochemistry Deparaffinized sections were incubated with citrate buffer solution (pH 6.0) and antigenicity was enhanced using an antigen-retrieval protocol in an autoclave (121°C, 1 min). Nonspecific binding was blocked by treating sections with 1% (w/v) bovine serum albumin (BSA) in 10 mM phosphate-buffered saline (PBS; pH 7.4). After blocking, sections were incubated with primary antibodies overnight at 4°C. The primary antibodies employed in this study were rabbit polyclonal antibodies raised against murine CK5 (Covance, Berkeley, CA, USA; 1:1000 dilution in PBS), CK8 (Progen, Heidelberg, Germany; diluted 1:100 in PBS) or CK14 (Covance; 1:1000 dilution in PBS). After washing with PBS, sections were incubated with biotinylated secondary antibodies raised against rabbit Ig (DAKO, Glostrup, Denmark) for 30 min at room temperature. After the sections were rinsed with PBS, they were incubated with Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) reagents, for signal enhancement, according to the manufacturer’s instructions, and the reaction was visualized with 0.05% (w/v) 3,3′-diaminobenzidine tetrahydrochloride (DAB) and 0.03% (v/v) H2O2 in 50 mM Tris buffer, pH 7.6 for 1–5 min. Sections were rinsed in tap water, counterstained with hematox-
Double-immunofluorescence labeling To further examine cells that were positive for CK5, CK8, and/or CK14 immunostaining, the epididymal preparations from three mice were double-labeled. Deparaffinized, rehydrated tissue sections were subjected to antigen retrieval, blocking, and primary antibody incubation as described above. After washing with PBS, tissue sections were incubated with Alexa Fluor® 568-labeled secondary antibody (Molecular Probes, Eugene, OR, USA) for 30 min at room temperature, and washed again. Double-labeling was achieved by subsequent incubation with anti-p63 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200 dilution in PBS), ZO-1 antibody (Invitrogen, Camarillo, CA, USA; 1:100 dilution in PBS) or GS-IB4 lectin (Vector Laboratories; 40 μg/ml), and then FITC-labeled secondary antibodies against rabbit Ig (Jackson ImmunoResearch, West Grove, PA, USA). After washing, slides were counterstained with Hoechst 33258 (Sigma-Aldrich, St Louis, MO, USA), mounted with a Vectashield (Vector Laboratories), and examined using a BX51 fluorescence microscope (Olympus, Japan). The control specimens were prepared by omission of the primary antibodies. Results CK5 and CK14 are markers of epididymal basal cells In adult mice, CK5 and CK14 were exclusively expressed in the cytoplasm of oval or flat-shaped cells, which are located at the base of the epididymal epithelium, below other epithelial cell types (Fig. 2A, B). These CK5–CK14-positive cells were identified as basal cells by colocalization with p63 (Fig. 2C), which is a marker of epididymal basal cells in mice14,17) and rats18). By contrast, CK8 was expressed in all epithelial cells, including principal, clear, and basal cells (data not shown). No positive staining could be detected when primary antibody was omitted (data not shown). To estimate the number of basal cells positive for CK5 and CK14 in each cross-section, ≥ 100 epididymal tubule cross-sections were assessed in each region. The basal cells were significantly abundant in region I, which is the initial segment of the epididymis (Fig. 3). In region I, basal cells often showed a long, narrow, cytoplasmic extension that infiltrated between other epithelial cells toward the lumen (Fig. 2D, E). We designated these cells the flask-shaped basal cells. To determine whether the flask-shaped basal cells can cross the tight-junction barrier, double labeling for GS-IB4 lectin, which is a marker of basal cells19,20), and tight junction protein ZO-121), was performed on mouse epididymis. Fig. 2F
Epididymal basal cells during mouse development
Fig. 2.
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Expression of CK5 and CK14 in basal cells of the adult mouse epididymis. A: Immunostaining with CK14. B: Immunostaining with CK5. C: Double immunofluorescence imaging with CK14 (red) and the basal cell nuclear marker p63 (green), showing that p63-positive cells also express CK14. D: Immunostaining with CK5 in region I. E: High power view of immunostaining with CK5. F: A long, slender projection of a GS-IB4 lectin-positive cell (red) penetrates the ZO-1-positive tight junction (green), to project into the lumen (arrowhead) of region II. Bars = 50 μm for A, B, C, and D. Bars = 20 μm for E and F.
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Statistical analysis of flask-shaped basal cells A statistical analysis was performed to determine the number of basal cells with extending projections (flaskshaped basal cells). A projection was defined as a region located above the nuclei of adjacent epithelial cells. Differences in the numbers of flask-shaped cells between the five segments of the developing epididymis were distinguishable in 3-week-old mice. The frequency of the flask-shaped basal cells was higher in region I than in other regions (Fig. 5). Discussion
Fig. 3.
Quantification of the number of basal cells in different regions of the adult mouse epididymis. The left axis shows the mean number of basal cells per 0.1 mm of the perimeter of the tubules. Data are expressed as mean ± SEM *p < 0.005.
clearly shows a flask-shaped basal cell extending its body projection to cross the ZO1-labeled tight junction barrier and toward the lumen. Having determined that CK5 and CK14 are specific markers of basal cells in the mouse epididymis, we then followed the appearance and morphology of these cells during postnatal development in epididymal sections. Immunolocalization of epididymal basal cells during mouse development In the epididymis of mice aged 0–1 week, no histological differences could be found between columnar epithelial cells. The first appearance of basal cells, identified by positive labeling for CK5 and CK14, was observed in some columnar cells among immature epithelial cells in the epididymal duct of mice aged 1 week. No CK5 or CK14 immunostaining was observed in the epididymis of newborn mice (Fig. 4A, B). The reactivity with CK5 and CK14 immunohistochemistry became progressively stronger in some columnar cells throughout the epididymal epithelium in mice aged 2–3 weeks (Fig. 4C). In mice aged 3 weeks, the reactivity was strong in both dome-shaped and flask-shaped basal cells in regions IV and V, and was moderate in regions I–III. In mice aged ≥ 4 weeks, strong reactivity was observed in basal cells of all epididymal regions (Fig. 4D). The expression patterns of CK5 and CK14 in epididymal cells at different stages of development and in different anatomical regions are summarized in Table 1.
In the present study, we determined the immunolocalization of CK5 and CK14 in epididymal epithelium during postnatal development of the mouse, and identified that CK5 and CK14 were markers of differentiation of epididymal basal cells. These findings are consistent with those of studies carried out using rats10,13,18). We also demonstrated temporal and spatial differences in CK5 and CK14 expression with age and between regions, which may reflect differences in the functions of basal cells during postnatal development. Similar changes, associated with the development and differentiation of epididymal basal cells, have been observed in our previous studies on the structure of cellular sugar chains19, 20). Our immunohistochemical study revealed that the expression pattern of CK5 and CK14 in the developing epididymis attained adult characteristics around 4 weeks after birth. This observation suggests that functional differentiation (maturation) of basal cells is complete at this stage, which is just before spermatozoa begin to reach the epididymis. A similar phenomenon has been reported in previous studies6, 20, 22). Concerning the origin of epididymal basal cells, it has been proposed that they originate either from extratubular tissue23), or from immature columnar epithelial cells24). In this study, we found that basal cells positive for CK5 and CK14 first appeared in columnar epithelial cells consisting of immature epididymis, 1 week after birth. Furthermore, no obvious morphological difference was observed between columnar epithelial cells at this stage. Thus, in agreement with Shum and colleagues18), we support the idea that basal cells originate from columnar epithelial cells in the immature epididymis. However, in order to determine the origin of basal cells, it is required to identify that such immature epithelial cells have the capacity to differentiate into basal cells. Interestingly, we found two types of basal cells— dome-shaped and flask-shaped—in mice aged ≥ 3 weeks. We also showed that flask-shaped basal cells extend cytoplasmic projections that cross the tight-junction barrier and reach into the lumen. Shum and colleagues10,13) reported that, in rat epididymis, such flask-shaped basal
Epididymal basal cells during mouse development
Fig. 4.
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Immunostaining for CK14 in the proximal region of the postnatal mouse epididymis. A: No positive reaction is seen in immature epididymis of mice aged 0 week. B: Weak immunoreaction is seen in columnar epithelial cells (arrows) of mice aged 1 week. C: Moderate immunoreaction is seen in columnar or oval-shaped epithelial cells (arrows) of mice aged 2 weeks. D: Strong immunoreaction is seen in oval-shaped epithelial cells (arrows) of mice aged 4 weeks. Bars = 50 μm
Table 1. Expression of CK5 and CK14 in different regions of epididymal epithelium during postnatal development of the mouse age
region 0w 1w 2w 3w 4w 5w 6w 9w 13w
I
II
III
IV
V
– +/– + + ++ ++ ++ ++ ++
– +/– + + ++ ++ ++ ++ ++
– +/– + + ++ ++ ++ ++ ++
– +/– + ++ ++ ++ ++ ++ ++
– +/– + ++ ++ ++ ++ ++ ++
–; negative, +/–: weak positive, +; moderate positive, ++; strong positive
cells express Type II angiotensin receptor, suggesting that lumen-reaching basal cells are able to monitor the luminal fluid and regulate the functions of adjacent epithelial cells. However, receptors for luminal sensing have not yet been identified in mouse epididymis. Furthermore, in accordance with a recent study15), we found that many flask-shaped basal cells exist in the proximal region (the initial segment, or region I) of mouse epididymis. By contrast, flask-shaped basal cells in rats
were mainly observed in the distal epididymal regions (the distal corpus and proximal cauda)13). These discrepancies may result from functional differences between regions of the epididymis in different species. In conclusion, the results of our study indicate that, in the postnatal development of the mouse epididymis, CK5 and CK14 could be useful markers of the differentiation of epididymal basal cells, which originate from immature columnar epithelial cells 1 week after birth. The basal
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Fig. 5.
Quantification of the percentage of flask-shaped basal cells detected in different regions of the epididymis during postnatal development. The left axis shows the percentage of basal cells per 0.1 mm of the perimeter of the tubules.
cells are of two types, dome-shaped and flask-shaped, and the latter are mainly situated in the initial segment of the mature epididymis.
9) 10)
Acknowledgements
11)
This work was supported by JSPS KAKENHI Grant Numbers 22590175 and 25460246. 12)
References 1) Robaire B, Hinton BT, Orgebin-Crist MC: The epididymis. In: Neill JD, ed, Knobil and Neill’s Physiology of Reproduction, 3rd ed. New York, Elsevier 2006; 1071–1148. 2) Bedford JM: Maturation, transport and fate of spermatozoa in the epididymis. In: Hamilton DW, Greep RO, eds, Handbook of Physiology. Washington DC, American Physiology Society 1975; 303–317. 3) Yoshinaga K and Toshimori K: Organization and modifications of sperm acrosomal molecules during spermatogenesis and epididymal maturation. Microsc Res Techniq 2003; 61:39-45. 4) Takano H: Qualitative and quantitative histology and histogenesis of the mouse epididymis, with special emphasis on the regional difference. Kaibougaku Zasshi 1980; 55:573–587 (in Japanese). 5) Abou-Haila A, Fain-Maurel MA: Postnatal differentiation of the enzyme activity of the mouse epididymis. J Androl 1985; 8:441– 458. 6) Bendahmane M, Abou-Haila A: Characterization of glycoconjugates in the epididymal epithelium and luminal fluid during postnatal development of the mouse. Cell Tissue Res 1997; 287:611– 619. 7) Shum WW, Da Silva N, Brown D, Breton S: Regulation of luminal acidification in the male reproductive tract via cell-cell crosstalk. J Exp Biol 2009; 212:1753–1761. 8) Hermo L, Adamali HI, Andonian S: Immunolocalization of CA II
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and H+V-ATPase in epithelial cells of the mouse and rat epididymis. J Androl 2000; 21:376–391. Robaire B, Viger RS: Regulation of epididymal epithelial cell functions. Biol Reprod 1995; 52:226–236. Shum WW, Ruan YC, Da Silva N, Breton S: Establishment of cell-cell cross talk in the epididymis: control of luminal acidification. J Androl 2011; 32:576–586. Seiler P, Wenzel I, Wagenfeld A, Yeung CH, Nieschlag E, Cooper TG: The appearance of basal cells in the developing murine epididymis and their temporal expression of macrophage antigens. Int J Androl 1998; 21:217–226. Yeung CH, Nashan D, Sorg C, Oberpenning F, Schulze H, Nieschlag E, Cooper TG: Basal cells of the human epididymis– antigenic and ultrastructural similarities to tissue-fixed macrophages. Biol Reprod 1994; 50:917–926. Shum WW, Da Silva N, McKee M, Smith PJ, Brown D, Breton S: Transepithelial projections from basal cells are luminal sensors in pseudostratified epithelia. Cell 2008; 135:1108–1117. Murashima A, Miyagawa S, Ogino Y, Nishida-Fukuda H, Araki K, Matsumoto T, Kaneko T, Yoshinaga K, Yamamura K, Kurita T, Kato S, Anne M. Moon, Yamada G: Essential roles of androgen signaling in Wolffian duct stabilization and epididymal cell differentiation. Endocrinology 2011; 152:1640–1651. Shum WW, Smith TB, Cortez-Retamozo V, Grigoryeva LS, Roy JW, Hill E, Pittet MJ, Breton S, Da Silva N: Epithelial basal cells are distinct from dendritic cells and macrophages in the mouse epididymis. Biol Reprod 2014; 90:1–10. Bendahmane M, Abou-Haila A: Synthesis, characterization and hormonal regulation of epididymal proteins during postnatal development of the mouse. Differentiation 1994; 55:119–125. Hayashi T, Yoshinaga A, Ohno R, Ishii N, Kamata S, Yamada T: Expression of the p63 and notch signaling systems in sat testes during postnatal development: comparison with their expression levels in the epididymis and vas deferens. J Androl 2004; 25:692– 698. Shum WW, Hill E, Brown D, Breton S: Plasticity of basal cells during postnatal development in the rat epididymis. Reproduction 2013; 146:455–469. Tajiri S, Fukui T, Sawaguchi A, Yoshinaga K: Cell- and region-specific expression of sugar chains in the mouse epidid-
Epididymal basal cells during mouse development ymal epithelium using lectin histochemistry combined with immunohistochemistry. Okajimas Folia Anat Jpn 2012; 88:141–146. 20) Tajiri S, Fukui T, Sawaguchi A, Yoshinaga K: Changes in lectinbinding sites on epididymal cells during postnatal development of the mouse. Okajimas Folia Anat Jpn 2012; 88:153–157. 21) Park CJ, Lee JE, Oh YS, Shim S, Kim DM, Park NC, Park HJ, Gye MC: Postnatal changes in the expression of claudin-11 in the testes and excurrent ducts of the domestic rabbit (Oryctolagus cuniculus domesticus). J Androl 2011; 32:295–306.
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22) Veri JP, Hermo L & Robaire B: Immunocytochemical localization of the Yf subunit of glutathione S-transferase P shows regional variation in the staining of epithelial cells of the testis, efferent ducts, and epididymis of the male rat. J Androl 1993; 14:23–44. 23) Holschbach C, Cooper TG: A possible extratubular origin of epididymal basal cells in mice. Reproduction 2002; 123:517–525. 24) Sun EL, Flickinger CJ: Proliferative activity in the rat epididymis during postnatal development. Anat Rec 1982; 203:273–284.
(Original)
Cytokeratin localization and basal cell differentiation in the epididymal epithelium during postnatal development of the mouse By
Ayaka YOSHIDA, Kana MURAKAMI, Kentaro SAKUDA, and Kazuya YOSHINAGA Department of Anatomy and Cell Biology, Graduate School of Health Sciences, Kumamoto University, 4-24-1 Kuhonji, Kumamoto 862-0975, Japan – Received for Publication, January 19, 2015 – Key Words:
basal cells, cytokeratins, differentiation, immunohistochemistry, mouse epididymis
Summary: The epididymis is a male genital organ that has plays various functions, including sperm concentration, maturation, and storage. The epididymal epithelium consists of principal cells, clear cells, and basal cells. To comprehensively understand the occurrence and morphological differentiation of basal cells, we examined the expression and localization of cytokeratins (CKs) in the epididymal epithelium during postnatal development of the mouse. Immunohistochemical staining showed that, in adult mice, CK5 and CK14 were exclusively expressed in the cytoplasm of basal cells. During postnatal development, basal cells that stained positive for CK5 and CK14 first appeared in immature columnar epithelial cells in mice aged 1 week. The immunoreactivity became progressively stronger in mice aged 2–3 weeks. In mice aged 3 weeks, the immunoreactivity was strong in regions IV and V. In mice aged ≥ 4 weeks, strong immunoreactivity was observed in all epididymal regions. CK5 and CK14 could be useful markers of differentiation in epididymal basal cells. These basal cells originate from immature columnar epithelial cells and are of two types—dome-shaped and flask-shaped—. The flask-shaped cells are mainly located in the initial segment of the mouse epididymis.
Introduction The epididymis is a male genital organ that has a variety of functions, including concentration, maturation, protection, and storage of sperm1). Spermatozoa are formed in the testes and become functionally mature during their passage through the epididymal ducts, acquiring motility and a fertilizing capacity via biochemical and physiological changes1–3). The mouse epididymal duct can be divided into five regions—I (initial segment), II, III, IV, and V, according to the morphological properties of the epithelial cells in each region4) (Fig. 1). The epididymal epithelium consists mainly of principal cells, clear cells, and basal cells5–7). Functionally, it is known that principal cells contribute to absorption of luminal fluid and secretion of glycoproteins, and clear cells contribute to acidification of the lumen by secreting protons7–10). Although basal cells have been proposed to have potential roles as stem cells and sensor cells for monitoring the luminal environment11–13), their actual functions have not yet been determined.
Cytokeratins (CKs) comprise a large family of cytoskeletal intermediate-filament proteins, and the composition of CKs, with different structural components, varies according to the type or developmental stage of epithelial cells. Thus, CKs are, clinically, used as markers to diagnose epithelial tumors and to monitor the response to treatments. CKs are known to be expressed and localized in epididymal epithelial cells of mice14,15); however, little is known about the relationship between cytokeratin expression and the histological appearance and differentiation stage of basal cells. The present study was performed to elucidate the occurrence and localization of CKs in epididymal epithelium during postnatal development of the mouse, and to complement the few previous reports of the morphological differentiation of murine epididymal basal cells5, 14–16).
Corresponding author: Kazuya Yoshinaga, Department of Anatomy and Cell Biology, Graduate School of Health Sciences, Kumamoto University, 4-24-1 Kuhonji, Kumamoto 862-0975, Japan. E-mail: [email protected]
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ylin, progressively dehydrated, cleared, and mounted. Negative controls were obtained by omission of the primary antibodies.
Fig. 1.
Illustration of mouse epididymal regions. The adult mouse epididymis can be morphologically divided into five regions (I– V).
Materials and Methods Animals and tissue preparations Male C57BL/6 mice, differing in age by 1-week intervals from birth (week 0) to 13 weeks of age, were purchased from Japan SLC (Hamamatsu, Japan). The experiments using laboratory mice were approved by the Committee on Animal Research at Kumamoto University. At least three animals of each age were used. Each epididymis was fixed overnight at 4°C by perfusion with, or immersion in, 4% paraformaldehyde, embedded in paraffin, and 3- to 4-μm-thick sections were prepared. Immunohistochemistry Deparaffinized sections were incubated with citrate buffer solution (pH 6.0) and antigenicity was enhanced using an antigen-retrieval protocol in an autoclave (121°C, 1 min). Nonspecific binding was blocked by treating sections with 1% (w/v) bovine serum albumin (BSA) in 10 mM phosphate-buffered saline (PBS; pH 7.4). After blocking, sections were incubated with primary antibodies overnight at 4°C. The primary antibodies employed in this study were rabbit polyclonal antibodies raised against murine CK5 (Covance, Berkeley, CA, USA; 1:1000 dilution in PBS), CK8 (Progen, Heidelberg, Germany; diluted 1:100 in PBS) or CK14 (Covance; 1:1000 dilution in PBS). After washing with PBS, sections were incubated with biotinylated secondary antibodies raised against rabbit Ig (DAKO, Glostrup, Denmark) for 30 min at room temperature. After the sections were rinsed with PBS, they were incubated with Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) reagents, for signal enhancement, according to the manufacturer’s instructions, and the reaction was visualized with 0.05% (w/v) 3,3′-diaminobenzidine tetrahydrochloride (DAB) and 0.03% (v/v) H2O2 in 50 mM Tris buffer, pH 7.6 for 1–5 min. Sections were rinsed in tap water, counterstained with hematox-
Double-immunofluorescence labeling To further examine cells that were positive for CK5, CK8, and/or CK14 immunostaining, the epididymal preparations from three mice were double-labeled. Deparaffinized, rehydrated tissue sections were subjected to antigen retrieval, blocking, and primary antibody incubation as described above. After washing with PBS, tissue sections were incubated with Alexa Fluor® 568-labeled secondary antibody (Molecular Probes, Eugene, OR, USA) for 30 min at room temperature, and washed again. Double-labeling was achieved by subsequent incubation with anti-p63 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200 dilution in PBS), ZO-1 antibody (Invitrogen, Camarillo, CA, USA; 1:100 dilution in PBS) or GS-IB4 lectin (Vector Laboratories; 40 μg/ml), and then FITC-labeled secondary antibodies against rabbit Ig (Jackson ImmunoResearch, West Grove, PA, USA). After washing, slides were counterstained with Hoechst 33258 (Sigma-Aldrich, St Louis, MO, USA), mounted with a Vectashield (Vector Laboratories), and examined using a BX51 fluorescence microscope (Olympus, Japan). The control specimens were prepared by omission of the primary antibodies. Results CK5 and CK14 are markers of epididymal basal cells In adult mice, CK5 and CK14 were exclusively expressed in the cytoplasm of oval or flat-shaped cells, which are located at the base of the epididymal epithelium, below other epithelial cell types (Fig. 2A, B). These CK5–CK14-positive cells were identified as basal cells by colocalization with p63 (Fig. 2C), which is a marker of epididymal basal cells in mice14,17) and rats18). By contrast, CK8 was expressed in all epithelial cells, including principal, clear, and basal cells (data not shown). No positive staining could be detected when primary antibody was omitted (data not shown). To estimate the number of basal cells positive for CK5 and CK14 in each cross-section, ≥ 100 epididymal tubule cross-sections were assessed in each region. The basal cells were significantly abundant in region I, which is the initial segment of the epididymis (Fig. 3). In region I, basal cells often showed a long, narrow, cytoplasmic extension that infiltrated between other epithelial cells toward the lumen (Fig. 2D, E). We designated these cells the flask-shaped basal cells. To determine whether the flask-shaped basal cells can cross the tight-junction barrier, double labeling for GS-IB4 lectin, which is a marker of basal cells19,20), and tight junction protein ZO-121), was performed on mouse epididymis. Fig. 2F
Epididymal basal cells during mouse development
Fig. 2.
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Expression of CK5 and CK14 in basal cells of the adult mouse epididymis. A: Immunostaining with CK14. B: Immunostaining with CK5. C: Double immunofluorescence imaging with CK14 (red) and the basal cell nuclear marker p63 (green), showing that p63-positive cells also express CK14. D: Immunostaining with CK5 in region I. E: High power view of immunostaining with CK5. F: A long, slender projection of a GS-IB4 lectin-positive cell (red) penetrates the ZO-1-positive tight junction (green), to project into the lumen (arrowhead) of region II. Bars = 50 μm for A, B, C, and D. Bars = 20 μm for E and F.
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Statistical analysis of flask-shaped basal cells A statistical analysis was performed to determine the number of basal cells with extending projections (flaskshaped basal cells). A projection was defined as a region located above the nuclei of adjacent epithelial cells. Differences in the numbers of flask-shaped cells between the five segments of the developing epididymis were distinguishable in 3-week-old mice. The frequency of the flask-shaped basal cells was higher in region I than in other regions (Fig. 5). Discussion
Fig. 3.
Quantification of the number of basal cells in different regions of the adult mouse epididymis. The left axis shows the mean number of basal cells per 0.1 mm of the perimeter of the tubules. Data are expressed as mean ± SEM *p < 0.005.
clearly shows a flask-shaped basal cell extending its body projection to cross the ZO1-labeled tight junction barrier and toward the lumen. Having determined that CK5 and CK14 are specific markers of basal cells in the mouse epididymis, we then followed the appearance and morphology of these cells during postnatal development in epididymal sections. Immunolocalization of epididymal basal cells during mouse development In the epididymis of mice aged 0–1 week, no histological differences could be found between columnar epithelial cells. The first appearance of basal cells, identified by positive labeling for CK5 and CK14, was observed in some columnar cells among immature epithelial cells in the epididymal duct of mice aged 1 week. No CK5 or CK14 immunostaining was observed in the epididymis of newborn mice (Fig. 4A, B). The reactivity with CK5 and CK14 immunohistochemistry became progressively stronger in some columnar cells throughout the epididymal epithelium in mice aged 2–3 weeks (Fig. 4C). In mice aged 3 weeks, the reactivity was strong in both dome-shaped and flask-shaped basal cells in regions IV and V, and was moderate in regions I–III. In mice aged ≥ 4 weeks, strong reactivity was observed in basal cells of all epididymal regions (Fig. 4D). The expression patterns of CK5 and CK14 in epididymal cells at different stages of development and in different anatomical regions are summarized in Table 1.
In the present study, we determined the immunolocalization of CK5 and CK14 in epididymal epithelium during postnatal development of the mouse, and identified that CK5 and CK14 were markers of differentiation of epididymal basal cells. These findings are consistent with those of studies carried out using rats10,13,18). We also demonstrated temporal and spatial differences in CK5 and CK14 expression with age and between regions, which may reflect differences in the functions of basal cells during postnatal development. Similar changes, associated with the development and differentiation of epididymal basal cells, have been observed in our previous studies on the structure of cellular sugar chains19, 20). Our immunohistochemical study revealed that the expression pattern of CK5 and CK14 in the developing epididymis attained adult characteristics around 4 weeks after birth. This observation suggests that functional differentiation (maturation) of basal cells is complete at this stage, which is just before spermatozoa begin to reach the epididymis. A similar phenomenon has been reported in previous studies6, 20, 22). Concerning the origin of epididymal basal cells, it has been proposed that they originate either from extratubular tissue23), or from immature columnar epithelial cells24). In this study, we found that basal cells positive for CK5 and CK14 first appeared in columnar epithelial cells consisting of immature epididymis, 1 week after birth. Furthermore, no obvious morphological difference was observed between columnar epithelial cells at this stage. Thus, in agreement with Shum and colleagues18), we support the idea that basal cells originate from columnar epithelial cells in the immature epididymis. However, in order to determine the origin of basal cells, it is required to identify that such immature epithelial cells have the capacity to differentiate into basal cells. Interestingly, we found two types of basal cells— dome-shaped and flask-shaped—in mice aged ≥ 3 weeks. We also showed that flask-shaped basal cells extend cytoplasmic projections that cross the tight-junction barrier and reach into the lumen. Shum and colleagues10,13) reported that, in rat epididymis, such flask-shaped basal
Epididymal basal cells during mouse development
Fig. 4.
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Immunostaining for CK14 in the proximal region of the postnatal mouse epididymis. A: No positive reaction is seen in immature epididymis of mice aged 0 week. B: Weak immunoreaction is seen in columnar epithelial cells (arrows) of mice aged 1 week. C: Moderate immunoreaction is seen in columnar or oval-shaped epithelial cells (arrows) of mice aged 2 weeks. D: Strong immunoreaction is seen in oval-shaped epithelial cells (arrows) of mice aged 4 weeks. Bars = 50 μm
Table 1. Expression of CK5 and CK14 in different regions of epididymal epithelium during postnatal development of the mouse age
region 0w 1w 2w 3w 4w 5w 6w 9w 13w
I
II
III
IV
V
– +/– + + ++ ++ ++ ++ ++
– +/– + + ++ ++ ++ ++ ++
– +/– + + ++ ++ ++ ++ ++
– +/– + ++ ++ ++ ++ ++ ++
– +/– + ++ ++ ++ ++ ++ ++
–; negative, +/–: weak positive, +; moderate positive, ++; strong positive
cells express Type II angiotensin receptor, suggesting that lumen-reaching basal cells are able to monitor the luminal fluid and regulate the functions of adjacent epithelial cells. However, receptors for luminal sensing have not yet been identified in mouse epididymis. Furthermore, in accordance with a recent study15), we found that many flask-shaped basal cells exist in the proximal region (the initial segment, or region I) of mouse epididymis. By contrast, flask-shaped basal cells in rats
were mainly observed in the distal epididymal regions (the distal corpus and proximal cauda)13). These discrepancies may result from functional differences between regions of the epididymis in different species. In conclusion, the results of our study indicate that, in the postnatal development of the mouse epididymis, CK5 and CK14 could be useful markers of the differentiation of epididymal basal cells, which originate from immature columnar epithelial cells 1 week after birth. The basal
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Fig. 5.
Quantification of the percentage of flask-shaped basal cells detected in different regions of the epididymis during postnatal development. The left axis shows the percentage of basal cells per 0.1 mm of the perimeter of the tubules.
cells are of two types, dome-shaped and flask-shaped, and the latter are mainly situated in the initial segment of the mature epididymis.
9) 10)
Acknowledgements
11)
This work was supported by JSPS KAKENHI Grant Numbers 22590175 and 25460246. 12)
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