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Primary cilia in the basal cells of equine epididymis: A serendipitous finding
Tissue and Cell 45 (2013) 140–144
Contents lists available at SciVerse ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Primary cilia in the basal cells of equine epididymis: A serendipitous finding Silvana Arrighi ∗ Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, Via Trentacoste 2, I-20134 Milan, Italy
a r t i c l e
i n f o
Article history: Received 26 June 2012 Received in revised form 10 October 2012 Accepted 11 October 2012 Available online 22 November 2012 Keywords: Primary cilia Epididymis Basal cells Electron microscopy (EM) Equidae
a b s t r a c t Occurrence of a solitary cilium was an unexpected discovery while studying the ultrastructure of epididymal epithelium in equidae. Primary cilia were detected in epididymal basal cells of all individuals of the equines studied – horses, donkey and mules – independently from age and tract of the duct, emerging from the basal cell surface and insinuating into the intercellular spaces. More rarely solitary cilia occurred also at the luminal surface of the principal cells. The ciliary apparatus was constituted by a structurally typical basal body continuous with the finger-like ciliary shaft extending from the cell surface, and an adjacent centriole oriented at right angles to the basal body. The cilium was structured as the typical primary, non-motile cilia found in many mammalian cells, having a 9 + 0 microtubular pattern. The basal diplosome was randomly associated with other cellular organelles including the Golgi complex, the endoplasmic reticulum, the microfilament network, the plasma membrane, vesicles and pits. Primary ciliogenesis is a new and unexpected finding in the epididymal epithelium. A monitoring role of luminal factors and extracellular liquids might be attributed to this organelle, likely acting as chemical receptor of the luminal environment, thus modulating the epithelial function by a cell-to-cell crosstalk involving the entire epithelium. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction More than thirty-five years ago David W. Hamilton, expert on epididymal structure and function, had to affirm that “epididymal basal cells are an enigma” (Hamilton, 1975). At that time, basal cells were attributed a stem function in the epithelium renewal (Hamilton, 1975; Ramos and Dym, 1977). Soon later, the opposite view, that principal cells might give rise to basal cells, was proposed by Sun and Flickinger (1982), a hypothesis not compatible with the low mitotic activity in the epididymis (Sujarit and Jones, 1991). A simple mechanical role in giving stability to the epithelium was also suggested, owing to the presence of filament bundles and membrane infoldings (Abe and Takano, 1989). A scavenger role was later proposed for equine epididymal basal cells, acting as macrophages having the task to remove from the epithelium waste matter derived from the endocytotic and spermatophagic activity of the principal cells (Arrighi et al., 1991, 1993, 1994). In the same years, macrophage-like qualities of basal cells were hypothesized (Yeung et al., 1994; Seiler et al., 1998) together with their possible extratubular origin from circulating monocytes (Holschbach and Cooper, 2002).
∗ Correspondence address: Department of Health, Animal Science and Food Safety, Laboratory of Anatomy – 2, Via Trentacoste, I-20134 Milano, Italy. Tel.: +39 0250315741; fax: +39 0250315746. E-mail address: [email protected] 0040-8166/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tice.2012.10.003
The most recent interpretation for the role of epididymal basal cells is that they might scan and sense the luminal environment of pseudostratified epithelia and modulate epithelial function by a mechanism involving crosstalk with other epithelial cells (Shum et al., 2008). This might be a paradigm shared by all basal cells residing in the so-called pseudostratified epithelia. The presence of a primary cilium emerging from the basal cell surface and insinuating into intercellular spaces was accidently found during ultrastructural studies on the epididymis in equidae. Although serendipitous in nature, the discovery of primary cilia in epididymal basal cells is a convincing finding, which completes the information on the likely functional roles most recently attributed to this enigmatic cell type. 2. Materials and methods Specimens were collected at slaughtering or at castration from healthy pubertal horses (n = 3), donkeys (n = 3) and mules (n = 3) aged from 4 to 15 years. Samples were taken from the different epididymis regions, which are macroscopically recognizable: two successive samples were collected at the caput level, three at the corpus level and one in the cauda (Nicander, 1958). Pieces intended for histological examination were fixed in formalin 10% for 24 h at 4 ◦ C. After fixation, fragments were dehydrated in a graded series of ethanol and embedded in paraffin. Serial sections were cut at 4 m thickness, de-waxed, and stained with routine hematoxylin and eosin (H&E).
S. Arrighi / Tissue and Cell 45 (2013) 140–144
Pieces intended for electron microscopy were trimmed and fixed in 2.5% glutaraldeyde in 0.1 M phosphate buffer at pH 7.4 for 3 h, then post-fixed in 1% OsO4 in the same buffer for 2 h. Fragments were then dehydrated through a graded series of ethanol and embedded in EPON 812. Ultrathin sections were obtained by an ultramicrotome (Ultratome IV, LKB, Sweden) collected on uncoated copper grids and counterstained with uranyl acetate and lead citrate, then examined and photographed under a Zeiss EM 109.
3. Results Epididymal epithelium in equidae was composed of principal cells, apical cells and basal cell. As in many other mammals, principal and apical cells were columnar whereas basal cells were roundish and rested on the basal lamina without reaching the lumen. Migrating elements such as lymphocytes and macrophages were also seen. LM pictures from the corpus (Fig. 1a) and cauda (Fig. 1b) of the horse epididymis are shown to orientate the reader. Basal cells were apparently more numerous and large in the corpus region. Ultrastructure of the epithelium lining the epididymis in the horse, donkey and mule has been previously described in detail (Romanello et al., 1985; Arrighi et al., 1991, 1993). A low magnification EM picture depicting the most basal region of the mule epididymal epithelium is shown in Fig. 1c. Morphology of the equine epididymal basal cell is typically characterized by the roundish shape, large, round and euchromatic nucleus and a cytoplasm poor of organelles (Fig. 1d). The cellular profile is characterized by cytoplasmic extensions and membrane digitations by which the basal cell connect to adjacent epithelial cells. The usual junctional devices were present, joining the basal cells to adjacent epithelial cells all along lateral cell surface. Between the abovementioned junctions, the facing cell membranes were separated from each other in such a way that a tiny space was often present between them (Figs. 1d and 2). The cell region facing the basal membrane was prevalently straight, connected to the basal lamina by hemidesmosomes (Fig. 2c). Frequently, micropinocytotic vesicles were present. In the cytoplasm, microfilament bundles were often prominent especially in the area surrounding the nucleus (Figs. 1 and 2c, d). Scattered profiles of endoplasmic reticulum, few mitochondria and a poorly developed Golgi apparatus were also present, without a precise localization within the cytoplasm. Residual bodies having a heterogeneous aspect were often present (Fig. 2a), together with lipid globules. The presence of solitary cilia was observed in the epididymal basal cells of all individuals studied, independently of species, age and epididymal tract considered (Figs. 1d and 2). Solitary cilia were more frequently observed in the basal cells of the corpus region. They originated from the basal cell surface, frequently extending in a tunnel-like invagination of the cell membrane and thereafter insinuating into the intercellular space (Fig. 2a, b and d). Rarely was the cilium followed for a relatively long distance among adjacent cells (Fig. 2a) whereas more often it disappeared from the plane of section after emerging straight from the cellular surface (Fig. 2b, d and e). The place where the cilium emerged was not fixed, and it never appeared facing the basement membrane. The ciliary apparatus was randomly associated with other cellular organelles including the Golgi complex, the endoplasmic reticulum, the microfilament network, the plasma membrane, vesicles and pits (Fig. 2b, arrows). The ciliary apparatus was constituted by a structurally typical basal body continuous with the finger-like ciliary shaft extending from the cell surface, and
141
by an adjacent centriole orientated at right angles to the basal body (Fig. 2c, d and e). The microtubular arrangement was typical of non-motile cilia, which have a 9 + 0 microtubular pattern. Nine peripherally arranged triads of microtubules were present whereas the central pair was lacking (Fig. 2e and f). In appropriate section accessory structures were seen, such as the basal plate (Fig. 2c, black arrow), alar sheet (Fig. 2d and e) and ciliary rootlets (Fig. 2c and e, white arrows). See Fig. 2 legend for further description. Much more rarely solitary cilia occurred at the luminal surface of the epididymal principal cells (Fig. 3). The ciliary shaft was directed into the lumen (Fig. 3a). Also in this case a 9 + 0 microtubular pattern was present (Fig. 3b).
4. Discussion During recent years intensive studies have been focused on the primary cilium projecting from the surface of vertebrate cells, thus transforming it from a poorly understood curiosity into a structure recognized for its importance in development, inherited human disease and cancer. Cilia and flagella are ancient structures that have evolved in organisms as diverse as protozoa and vertebrates. It is now known that cilia are essential during development for the response to developmental signals, and evidence is accumulating that the primary cilium is specialized for hedgehog signal transduction, acting via the evolutionarily conserved mechanism of intraflagellar transport (Goetz and Anderson, 2010). Moreover, human disorders – called ciliopathies – have been described, affecting diverse organ systems and deriving on underlying structural or functional abnormalities of primary cilia (Tobin and Beales, 2009). Apart from its involvement in differentiation and cell division, the primary cilium is known to be able to coordinate several essential cell signaling pathways, functioning as a dual sensor, mechanosensor and chemosensor (Singla and Reiter, 2006). The role of cilia in sensing the extracellular environment is best understood in the context of olfaction and photoreception. The innovative attribute of primary cilia as cellular antennae that sense a wide variety of signals might potentially take place in almost every vertebrate cell having a cell surface needing to be monitored, and a cross-talk to be performed. There has been a persistent literature on primary cilia from the early 1960s, summarized in part by Wheatley (2005). Actually, primary cilia have been reported in many cell types of all the embryonic origin and differentiated localization in the body (Satir and Christensen, 2007). Although meticulously reviewed up to recent years by Bowser and Wheatley in the Primary Cilium Resource Page, no mention exists of the presence of primary cilia in the epithelial cells of the mammalian epididymis. Neither the presence of primary cilia has ever been reported in the literature specifically directed to studies on epididymis, to the best of our knowledge. On the contrary the interest on the functions, and correlated morphology, of the epididymal basal cell is still topical. Remarkable studies published in the last five years are still wondering about the possible role of this enigmatic cells type in the manifold functions of the epididymal epithelium. According to the review by Cornwall (2009) basal cells – not accessing the luminal compartment – are in close association with the overlying principal cells and, by the presence of cytoplasmic extensions between principal cells may regulate its functions. Thus, basal cells are considered to regulate principal cell electrolyte transport by releasing paracrine factors. Moreover, cell–cell interactions within the epithelium can directly affect the luminal environment and ultimately sperm maturation. Recently, Shum et al. (2008) elegantly demonstrated that basal cells, previously believed to be
142
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Fig. 1. (a) and (b) LM micrographs of the horse epididymis. H&E staining. At the level of the corpus (a) the pseudostratified epithelium is build up by tall columnar principal cells with stereocilia and basally located, roundish basal cells which contact the basal lamina. At the level of the cauda (b) the epithelial thickness decreases and basal cells are smaller than in the corpus. (c) Low magnification EM micrograph of the epithelium lining the mule caput epididymis. Columnar principal cells (pc), basal cells (bc) and migrating lymphocytes (ly) can be seen. The basal cells rest on the basal membrane. They are roundish, have euchromatic nucleus and pale cytoplasm. (d) Basal cells in the epithelium lining the mule epididymis. Note the slightly indented cytoplasmic profile, the large and euchromatic nucleus and paucity of organelles. Microfilament bundles can be seen (arrows) around the nucleus. In the cytoplasm of the basal cell on the right a ciliary apparatus can be seen (white arrow). In the cytoplasm of neighbor principal cells large residual bodies are present mixed to other organelles.
restricted to the basal region of pseudostratified epithelia, send long and slender cytoplasmic projections infiltrating between other epithelial cells toward the lumen. The authors conclude that basal cells cross the blood/epididymis barrier to monitor luminal factors. The serendipitous finding of solitary cilia in the equine epididymis, mostly at the level of the corpus region, adds information to the studies aimed at elucidating the cellular mechanisms by which the epithelium responds to luminal stimuli. Also, micropinocytosis on the basal membrane facing the stroma and the blood vessels just underneath, might indicate movements of macromolecules. In this context, the presence of a primary cilium at the basal cell surface might constitute a monitoring
system for extracellular liquids and an indirect way to control the microenvironment. The lack of cilia protruding basally into the extra-tubular space might suggest that the putative chemosensor function would be sampling intra-epithelial, extracellular fluid but not extra-tubular fluid. Yet, another role stemming from the current observations might be put forward: as the basal cells with cilia also contain residual bodies, they might be phagocytes (Arrighi et al., 1991, 1993, 1994; Yeung et al., 1994; Seiler et al., 1998), so the sensors might be detecting antigens and directly affecting basal cell endocytosis. Much more sporadically, also during the ultrastructural study of the feline epididymis (Arrighi et al., 1986) solitary cilia were observed in the basal cells (unpublished observation). Although their occurrence was very rare and not linked to a
S. Arrighi / Tissue and Cell 45 (2013) 140–144
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Fig. 2. Different aspects of the solitary cilium and ciliary apparatus in the basal cells of the equine epididymal epithelium. (a) In the mule epididymal corpus a cilium can be seen, running in the intercellular space between two basal cells (arrows). The cytoplasm of the cell on the left contains many residual bodies (rb). (b) In the horse epididymal corpus the origin of a solitary cilium can be seen. The cilium extends in a tunnel-like invagination of the cell membrane, the ciliary pit, where endocytotic vesicles are present (arrows). (c) A couple of centrioles can be seen in the most basal rim of a basal cell in the donkey corpus epididymis, in the same cytoplasmic area where prominent filament bundles can be seen (thin arrows). The mother centriole differentiating into a ciliary basal body shows a basal plate which is indicated by the black arrow. The white arrow indicates a ciliary rootlet. Asterisks indicate the connection of the basal cell to the basal lamina. (d) Ciliary apparatus in a donkey epididymis basal cell. The primary cilium resides within a deep invagination that is continuous with the plasma membrane, the ciliary pit. The basal body resides at the base of the ciliary pit and is associated with its membrane via lateral appendages of pericentriolar material (arrows). A second centriole is present, orientated at right angles to the basal body. Mitochondria and RER can be seen in close vicinity to the cilium. (e) Diplosome in a basal cell of the horse epididymis. The ciliary shaft emerges from the mother centriole on the left, whose associated pericentriolar material can also be seen (curved arrow). The white arrow indicates a triangular-shaped ciliary rootlet extending from the daughter centriole into the cytoplasm. Prominent filament bundles are indicated by subtle arrows. (f) A typical 9 + 0 microtubular pattern is shown in a centriole localized in a basal cell of the horse epididymis, in close vicinity to an intercellular space (asterisks). Ciliary rootlets emerge from the centriole into the cytoplasm (white arrow).
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Fig. 3. Adluminal zone of principal cells of the epithelium lining the equine epididymis. (a) A ciliary shaft emerging from the mother centriole projects into the lumen of the donkey caput epididymidis. The daughter centriole shows the typical 9 + 0 microtubular pattern. (b) Typical 9 + 0 microtubular pattern is shown in a centriole localized in a principal cell of the mule cauda epididymidis. Peripheral triplets of microtubules are connected in the center by radial fibers. Ciliary rootlets emerge from the centriole into the cytoplasm (white arrow).
specific epididymal region, it indicated that this finding is possibly not limited to equidae. Sources of funding This study was supported by grants from Università degli Studi di Milano, Italy. References Abe, K., Takano, H., 1989. Cytological response of the principal cells in the initial segment of the epididymal duct to efferent duct cutting in mice. Arch. Histol. Cytol. 52, 321–326. Arrighi, S., Romanello, M.G., Domeneghini, C., 1986. Ultrastructural study on the epithelium lining ductus epididymis in adult cats (Felis catus). Arch. Biol. 97, 7–24. Arrighi, S., Romanello, M.G., Domeneghini, C., 1991. Morphological examination of epididymal epithelium in the mule in comparison with parental species (Equus asinus and Equus caballus). Histol. Histopathol. 6, 325–337. Arrighi, S., Romanello, M.G., Domeneghini, C., 1993. Ultrastructure of epididymal epithelium in Equus caballus. Ann. Anat. 175, 1–9. Arrighi, S., Romanello, M.G., Domeneghini, C., 1994. Ultrastructure of the epithelium that lines the ductuli efferentes in domestic equidae, with particular reference to spermatophagy. Acta Anat. 149, 174–184. Bowser, S.S., Wheatley, D.N. Primary Cilium Resource Page List of Cells. (www.bowserlab.org/primarycilia/cilialist.html). Cornwall, G.A., 2009. New insights into epididymal biology and function. Hum. Reprod. Update 15, 213–227. Goetz, S.C., Anderson, K.V., 2010. The primary cilium: a signalling centre during vertebrate development. Nat. Rev. Genet. 11, 331–344. Hamilton, D.W., 1975. Structure and function of the epithelium lining the ductuli efferentes, ductus epididymis and ductus deferens in the rat. In: Hamilton,
D.W., Creep, R.O. (Eds.), Handbook of Physiology. Section 7 Endocrinology. Male Reproductive System, vol. V. Am. Physiol. Soc., Washington DC, pp. 259–301. Holschbach, C., Cooper, T.G., 2002. A possible extratubular origin of epididymal basal cells in mice. Reproduction 123, 517–525. Nicander, L., 1958. Studies on the regional histology and cytochemistry of the ductus epididymis in stallion, rams and bulls. Acta Morphol. Neerl. Scand. 1, 337–362. Ramos Jr., A.S., Dym, M., 1977. Fine structure of the monkey epididymis. Am. J. Anat. 149, 501–531. Romanello, M.G., Arrighi, S., Domeneghini, C., Rizzotti, M., 1985. Histochemical and ultrastructural studies on the epithelium of the head of the epididymis in the donkey. Schweizer. Arch. Tierheilk. 127, 671–684. Satir, P., Christensen, S.T., 2007. Overview of structure and function of mammalian cilia. Annu. Rev. Physiol. 69, 377–400. Seiler, P., Wenzel, I., Wagenfeld, A., Yeung, C.-H., Nieschlag, E., Cooper, T.G., 1998. The appearance of basal cells in the developing murine epididymis and their temporal expression of macrophage antigens. Int. J. Androl. 21, 217–226. Shum, W.W., Da Silva, N., McKee, M., Smith, P.J., Brown, D., Breton, S., 2008. Transepithelial projections from basal cells are luminal sensors in pseudostratified epithelia. Cell 135, 1108–1117. Singla, V., Reiter, J.F., 2006. The primary cilium as the cell’s antenna: signaling at a sensory organelle. Science 313, 629–633. Sun, E.L., Flickinger, C.J., 1982. Proliferative activity in the rat epididymis during postnatal development. Anat. Rec. 203, 273–284. Sujarit, S., Jones, R.C., 1991. [3 H]Thymidine uptake by the epididymis, seminal vesicles and prostate gland during postnatal development of the rat. Reprod. Fertil. Dev. 3, 313–319. Tobin, J.L., Beales, P.L., 2009. The nonmotile ciliopathies. Genet. Med. 11, 386–402. Wheatley, D.N., 2005. Landmarks in the first hundred years of primary (9 + 0) cilium research. Cell Biol. Int. 29, 333–339. Yeung, C.H., Nashan, D., Cooper, T.G., Sorg, C., Oberpenning, F., Schulze, H., Nieschlag, E., 1994. Basal cells of the human epididymis – antigenic and ultrastructural similarities to tissue-fixed macrophages. Biol. Reprod. 50, 917–926.
Contents lists available at SciVerse ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Primary cilia in the basal cells of equine epididymis: A serendipitous finding Silvana Arrighi ∗ Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, Via Trentacoste 2, I-20134 Milan, Italy
a r t i c l e
i n f o
Article history: Received 26 June 2012 Received in revised form 10 October 2012 Accepted 11 October 2012 Available online 22 November 2012 Keywords: Primary cilia Epididymis Basal cells Electron microscopy (EM) Equidae
a b s t r a c t Occurrence of a solitary cilium was an unexpected discovery while studying the ultrastructure of epididymal epithelium in equidae. Primary cilia were detected in epididymal basal cells of all individuals of the equines studied – horses, donkey and mules – independently from age and tract of the duct, emerging from the basal cell surface and insinuating into the intercellular spaces. More rarely solitary cilia occurred also at the luminal surface of the principal cells. The ciliary apparatus was constituted by a structurally typical basal body continuous with the finger-like ciliary shaft extending from the cell surface, and an adjacent centriole oriented at right angles to the basal body. The cilium was structured as the typical primary, non-motile cilia found in many mammalian cells, having a 9 + 0 microtubular pattern. The basal diplosome was randomly associated with other cellular organelles including the Golgi complex, the endoplasmic reticulum, the microfilament network, the plasma membrane, vesicles and pits. Primary ciliogenesis is a new and unexpected finding in the epididymal epithelium. A monitoring role of luminal factors and extracellular liquids might be attributed to this organelle, likely acting as chemical receptor of the luminal environment, thus modulating the epithelial function by a cell-to-cell crosstalk involving the entire epithelium. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction More than thirty-five years ago David W. Hamilton, expert on epididymal structure and function, had to affirm that “epididymal basal cells are an enigma” (Hamilton, 1975). At that time, basal cells were attributed a stem function in the epithelium renewal (Hamilton, 1975; Ramos and Dym, 1977). Soon later, the opposite view, that principal cells might give rise to basal cells, was proposed by Sun and Flickinger (1982), a hypothesis not compatible with the low mitotic activity in the epididymis (Sujarit and Jones, 1991). A simple mechanical role in giving stability to the epithelium was also suggested, owing to the presence of filament bundles and membrane infoldings (Abe and Takano, 1989). A scavenger role was later proposed for equine epididymal basal cells, acting as macrophages having the task to remove from the epithelium waste matter derived from the endocytotic and spermatophagic activity of the principal cells (Arrighi et al., 1991, 1993, 1994). In the same years, macrophage-like qualities of basal cells were hypothesized (Yeung et al., 1994; Seiler et al., 1998) together with their possible extratubular origin from circulating monocytes (Holschbach and Cooper, 2002).
∗ Correspondence address: Department of Health, Animal Science and Food Safety, Laboratory of Anatomy – 2, Via Trentacoste, I-20134 Milano, Italy. Tel.: +39 0250315741; fax: +39 0250315746. E-mail address: [email protected] 0040-8166/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tice.2012.10.003
The most recent interpretation for the role of epididymal basal cells is that they might scan and sense the luminal environment of pseudostratified epithelia and modulate epithelial function by a mechanism involving crosstalk with other epithelial cells (Shum et al., 2008). This might be a paradigm shared by all basal cells residing in the so-called pseudostratified epithelia. The presence of a primary cilium emerging from the basal cell surface and insinuating into intercellular spaces was accidently found during ultrastructural studies on the epididymis in equidae. Although serendipitous in nature, the discovery of primary cilia in epididymal basal cells is a convincing finding, which completes the information on the likely functional roles most recently attributed to this enigmatic cell type. 2. Materials and methods Specimens were collected at slaughtering or at castration from healthy pubertal horses (n = 3), donkeys (n = 3) and mules (n = 3) aged from 4 to 15 years. Samples were taken from the different epididymis regions, which are macroscopically recognizable: two successive samples were collected at the caput level, three at the corpus level and one in the cauda (Nicander, 1958). Pieces intended for histological examination were fixed in formalin 10% for 24 h at 4 ◦ C. After fixation, fragments were dehydrated in a graded series of ethanol and embedded in paraffin. Serial sections were cut at 4 m thickness, de-waxed, and stained with routine hematoxylin and eosin (H&E).
S. Arrighi / Tissue and Cell 45 (2013) 140–144
Pieces intended for electron microscopy were trimmed and fixed in 2.5% glutaraldeyde in 0.1 M phosphate buffer at pH 7.4 for 3 h, then post-fixed in 1% OsO4 in the same buffer for 2 h. Fragments were then dehydrated through a graded series of ethanol and embedded in EPON 812. Ultrathin sections were obtained by an ultramicrotome (Ultratome IV, LKB, Sweden) collected on uncoated copper grids and counterstained with uranyl acetate and lead citrate, then examined and photographed under a Zeiss EM 109.
3. Results Epididymal epithelium in equidae was composed of principal cells, apical cells and basal cell. As in many other mammals, principal and apical cells were columnar whereas basal cells were roundish and rested on the basal lamina without reaching the lumen. Migrating elements such as lymphocytes and macrophages were also seen. LM pictures from the corpus (Fig. 1a) and cauda (Fig. 1b) of the horse epididymis are shown to orientate the reader. Basal cells were apparently more numerous and large in the corpus region. Ultrastructure of the epithelium lining the epididymis in the horse, donkey and mule has been previously described in detail (Romanello et al., 1985; Arrighi et al., 1991, 1993). A low magnification EM picture depicting the most basal region of the mule epididymal epithelium is shown in Fig. 1c. Morphology of the equine epididymal basal cell is typically characterized by the roundish shape, large, round and euchromatic nucleus and a cytoplasm poor of organelles (Fig. 1d). The cellular profile is characterized by cytoplasmic extensions and membrane digitations by which the basal cell connect to adjacent epithelial cells. The usual junctional devices were present, joining the basal cells to adjacent epithelial cells all along lateral cell surface. Between the abovementioned junctions, the facing cell membranes were separated from each other in such a way that a tiny space was often present between them (Figs. 1d and 2). The cell region facing the basal membrane was prevalently straight, connected to the basal lamina by hemidesmosomes (Fig. 2c). Frequently, micropinocytotic vesicles were present. In the cytoplasm, microfilament bundles were often prominent especially in the area surrounding the nucleus (Figs. 1 and 2c, d). Scattered profiles of endoplasmic reticulum, few mitochondria and a poorly developed Golgi apparatus were also present, without a precise localization within the cytoplasm. Residual bodies having a heterogeneous aspect were often present (Fig. 2a), together with lipid globules. The presence of solitary cilia was observed in the epididymal basal cells of all individuals studied, independently of species, age and epididymal tract considered (Figs. 1d and 2). Solitary cilia were more frequently observed in the basal cells of the corpus region. They originated from the basal cell surface, frequently extending in a tunnel-like invagination of the cell membrane and thereafter insinuating into the intercellular space (Fig. 2a, b and d). Rarely was the cilium followed for a relatively long distance among adjacent cells (Fig. 2a) whereas more often it disappeared from the plane of section after emerging straight from the cellular surface (Fig. 2b, d and e). The place where the cilium emerged was not fixed, and it never appeared facing the basement membrane. The ciliary apparatus was randomly associated with other cellular organelles including the Golgi complex, the endoplasmic reticulum, the microfilament network, the plasma membrane, vesicles and pits (Fig. 2b, arrows). The ciliary apparatus was constituted by a structurally typical basal body continuous with the finger-like ciliary shaft extending from the cell surface, and
141
by an adjacent centriole orientated at right angles to the basal body (Fig. 2c, d and e). The microtubular arrangement was typical of non-motile cilia, which have a 9 + 0 microtubular pattern. Nine peripherally arranged triads of microtubules were present whereas the central pair was lacking (Fig. 2e and f). In appropriate section accessory structures were seen, such as the basal plate (Fig. 2c, black arrow), alar sheet (Fig. 2d and e) and ciliary rootlets (Fig. 2c and e, white arrows). See Fig. 2 legend for further description. Much more rarely solitary cilia occurred at the luminal surface of the epididymal principal cells (Fig. 3). The ciliary shaft was directed into the lumen (Fig. 3a). Also in this case a 9 + 0 microtubular pattern was present (Fig. 3b).
4. Discussion During recent years intensive studies have been focused on the primary cilium projecting from the surface of vertebrate cells, thus transforming it from a poorly understood curiosity into a structure recognized for its importance in development, inherited human disease and cancer. Cilia and flagella are ancient structures that have evolved in organisms as diverse as protozoa and vertebrates. It is now known that cilia are essential during development for the response to developmental signals, and evidence is accumulating that the primary cilium is specialized for hedgehog signal transduction, acting via the evolutionarily conserved mechanism of intraflagellar transport (Goetz and Anderson, 2010). Moreover, human disorders – called ciliopathies – have been described, affecting diverse organ systems and deriving on underlying structural or functional abnormalities of primary cilia (Tobin and Beales, 2009). Apart from its involvement in differentiation and cell division, the primary cilium is known to be able to coordinate several essential cell signaling pathways, functioning as a dual sensor, mechanosensor and chemosensor (Singla and Reiter, 2006). The role of cilia in sensing the extracellular environment is best understood in the context of olfaction and photoreception. The innovative attribute of primary cilia as cellular antennae that sense a wide variety of signals might potentially take place in almost every vertebrate cell having a cell surface needing to be monitored, and a cross-talk to be performed. There has been a persistent literature on primary cilia from the early 1960s, summarized in part by Wheatley (2005). Actually, primary cilia have been reported in many cell types of all the embryonic origin and differentiated localization in the body (Satir and Christensen, 2007). Although meticulously reviewed up to recent years by Bowser and Wheatley in the Primary Cilium Resource Page, no mention exists of the presence of primary cilia in the epithelial cells of the mammalian epididymis. Neither the presence of primary cilia has ever been reported in the literature specifically directed to studies on epididymis, to the best of our knowledge. On the contrary the interest on the functions, and correlated morphology, of the epididymal basal cell is still topical. Remarkable studies published in the last five years are still wondering about the possible role of this enigmatic cells type in the manifold functions of the epididymal epithelium. According to the review by Cornwall (2009) basal cells – not accessing the luminal compartment – are in close association with the overlying principal cells and, by the presence of cytoplasmic extensions between principal cells may regulate its functions. Thus, basal cells are considered to regulate principal cell electrolyte transport by releasing paracrine factors. Moreover, cell–cell interactions within the epithelium can directly affect the luminal environment and ultimately sperm maturation. Recently, Shum et al. (2008) elegantly demonstrated that basal cells, previously believed to be
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Fig. 1. (a) and (b) LM micrographs of the horse epididymis. H&E staining. At the level of the corpus (a) the pseudostratified epithelium is build up by tall columnar principal cells with stereocilia and basally located, roundish basal cells which contact the basal lamina. At the level of the cauda (b) the epithelial thickness decreases and basal cells are smaller than in the corpus. (c) Low magnification EM micrograph of the epithelium lining the mule caput epididymis. Columnar principal cells (pc), basal cells (bc) and migrating lymphocytes (ly) can be seen. The basal cells rest on the basal membrane. They are roundish, have euchromatic nucleus and pale cytoplasm. (d) Basal cells in the epithelium lining the mule epididymis. Note the slightly indented cytoplasmic profile, the large and euchromatic nucleus and paucity of organelles. Microfilament bundles can be seen (arrows) around the nucleus. In the cytoplasm of the basal cell on the right a ciliary apparatus can be seen (white arrow). In the cytoplasm of neighbor principal cells large residual bodies are present mixed to other organelles.
restricted to the basal region of pseudostratified epithelia, send long and slender cytoplasmic projections infiltrating between other epithelial cells toward the lumen. The authors conclude that basal cells cross the blood/epididymis barrier to monitor luminal factors. The serendipitous finding of solitary cilia in the equine epididymis, mostly at the level of the corpus region, adds information to the studies aimed at elucidating the cellular mechanisms by which the epithelium responds to luminal stimuli. Also, micropinocytosis on the basal membrane facing the stroma and the blood vessels just underneath, might indicate movements of macromolecules. In this context, the presence of a primary cilium at the basal cell surface might constitute a monitoring
system for extracellular liquids and an indirect way to control the microenvironment. The lack of cilia protruding basally into the extra-tubular space might suggest that the putative chemosensor function would be sampling intra-epithelial, extracellular fluid but not extra-tubular fluid. Yet, another role stemming from the current observations might be put forward: as the basal cells with cilia also contain residual bodies, they might be phagocytes (Arrighi et al., 1991, 1993, 1994; Yeung et al., 1994; Seiler et al., 1998), so the sensors might be detecting antigens and directly affecting basal cell endocytosis. Much more sporadically, also during the ultrastructural study of the feline epididymis (Arrighi et al., 1986) solitary cilia were observed in the basal cells (unpublished observation). Although their occurrence was very rare and not linked to a
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Fig. 2. Different aspects of the solitary cilium and ciliary apparatus in the basal cells of the equine epididymal epithelium. (a) In the mule epididymal corpus a cilium can be seen, running in the intercellular space between two basal cells (arrows). The cytoplasm of the cell on the left contains many residual bodies (rb). (b) In the horse epididymal corpus the origin of a solitary cilium can be seen. The cilium extends in a tunnel-like invagination of the cell membrane, the ciliary pit, where endocytotic vesicles are present (arrows). (c) A couple of centrioles can be seen in the most basal rim of a basal cell in the donkey corpus epididymis, in the same cytoplasmic area where prominent filament bundles can be seen (thin arrows). The mother centriole differentiating into a ciliary basal body shows a basal plate which is indicated by the black arrow. The white arrow indicates a ciliary rootlet. Asterisks indicate the connection of the basal cell to the basal lamina. (d) Ciliary apparatus in a donkey epididymis basal cell. The primary cilium resides within a deep invagination that is continuous with the plasma membrane, the ciliary pit. The basal body resides at the base of the ciliary pit and is associated with its membrane via lateral appendages of pericentriolar material (arrows). A second centriole is present, orientated at right angles to the basal body. Mitochondria and RER can be seen in close vicinity to the cilium. (e) Diplosome in a basal cell of the horse epididymis. The ciliary shaft emerges from the mother centriole on the left, whose associated pericentriolar material can also be seen (curved arrow). The white arrow indicates a triangular-shaped ciliary rootlet extending from the daughter centriole into the cytoplasm. Prominent filament bundles are indicated by subtle arrows. (f) A typical 9 + 0 microtubular pattern is shown in a centriole localized in a basal cell of the horse epididymis, in close vicinity to an intercellular space (asterisks). Ciliary rootlets emerge from the centriole into the cytoplasm (white arrow).
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Fig. 3. Adluminal zone of principal cells of the epithelium lining the equine epididymis. (a) A ciliary shaft emerging from the mother centriole projects into the lumen of the donkey caput epididymidis. The daughter centriole shows the typical 9 + 0 microtubular pattern. (b) Typical 9 + 0 microtubular pattern is shown in a centriole localized in a principal cell of the mule cauda epididymidis. Peripheral triplets of microtubules are connected in the center by radial fibers. Ciliary rootlets emerge from the centriole into the cytoplasm (white arrow).
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