Peritubular Contractile Cells in Testis and Epididymis of the Dog, Canis lupus familiaris

Contractile cells surrounding the tubular system of the mammalian testis and epididymis are supposed to contribute to the initial transport of spermatozoa from the testis to epididymis. Testicular peritubular smooth muscle cells have been characterised in detail especially in rodents and humans. The aim of our study was to assess the distribution of peritubular contractile cells of the canine tubuli seminiferi, rete testis channels, ductuli efferentes, and ductus epididymidis by immunohistochemistry and transmission electron microscopy and to classify these cells with respect to their possible physiological function. The entire tubular system of the canine testis and epididymis is surrounded by contractile cells expressing smooth muscle actin, smooth muscle myosin and desmin, which are enveloped, at least partly, by a basal lamina. Some contractile cells of the tubuli seminiferi, rete channels, and efferent ducts and sometimes also single peritubular cells of the ductus epididymidis express vimentin. Contractile cells of seminiferous tubules and efferent ducts represent an intermediate cell type exhibiting characteristics of both smooth muscle cells (SMC) and myofibroblasts, those of rete channels stellate myofibroblasts, and those of the ductus epididymidis SMC. Differences in structure and arrangement of the contractile cells between seminiferous tubules, rete channels, efferent ducts, and ductus epididymidis suggest different functions. Myofibroblasts and contractile cells similar to them could be mainly responsible for the maintenance of an appropriate tissue turgor, whereas contraction of SMC of the ductus epididymidis might cause propulsion of spermatozoa by peristaltic waves. Myofibroblasts, smooth muscle cells, sperm transport, immunohistochemistry, electron microscopy Contractile cells in the peritubular tissue (lamina propria) of the mammalian testis are supposed to contribute to the initial transport of spermatozoa from the testis to the epididymis. In the lamina propria of the seminiferous tubules, contractile cells have been found in many mammalian species such as the rat, mouse, hamster, rabbit, cat, sheep, cattle, pig and human (Roosen-Runge 1951; Ross and Long 1966; Ross 1967; Böck et al. 1972; Bressler and Ross 1972; Bustos-Obregón and Holstein 1973; De Kretser et al. 1975; Hargrove et al. 1977; Wrobel et al. 1979; Maekawa et al. 1996). Similar cells surround the channels of the rete testis (Roosen-Runge and Holstein 1978; Hees et al. 1989). The structure, quantity, and layering of testicular contractile cells vary by species (Maekawa et al. 1996). In early studies, the cells were usually referred to as myoid cells without further classification (Ross and Long 1966; Dierichs and Wrobel 1973; Bustos-Obregón and Courot 1974). Contractile cells surrounding the seminiferous tubules of rodents have recently been analysed in great detail and are usually termed peritubular smooth muscle cells (for overview, see e.g. Maekawa et al. 1996; Tripiciano et al. 1999; Romano et al. 2005, and references therein), although they seem to differ considerably from other smooth muscle cells (Romano et al. 2005). In some species, such as cattle and humans, these cells share characteristics with fibroblasts and have therefore been termed myofibroblasts (Böck et al. 1972; Wrobel et al. 1979; Hees et al. 1989; Holstein et al. 1996; Maekawa et al. 1996). In boars, only cells of the ACTA VET. BRNO 2009, 78: 3–11; doi:10.2754/avb200978010003 Address for correspondence: K. Witter Institute of Histology and Embryology Department of Pathobiology University of Veterinary Medicine Vienna Veterinärplatz 1, 1210 Wien, Austria Phone: +43 1 25077 3403 Fax: +43 1 25077 3490 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm inner peritubular layer are contractile, whereas outer layers consist of fibroblasts (Pinart et al. 2001). Studies on contractile cells surrounding rete channels and efferent ducts of mammals are rather rare (Baumgarten et al. 1971; Viotto et al. 1993; Rodríguez et al. 1999; Devkota et al. 2006), although they can also be expected to play a considerable role in the sperm transport. Contractile cells of the lamina propria of the ductus epididymidis are generally characterised as smooth muscle cells (Baumgarten et al. 1971; Whittington et al. 2001; see also textbooks on human and veterinary histology). Myofibroblasts resemble smooth muscle cells (SMC) in their contractility and their cytoskeletal proteins, but fibroblasts in their biosynthetic phenotype and their response to the regulation operated by extracellular matrix and mechanical forces (Lefebvre et al. 1994). They are usually spindle-shaped or stellate with long cytoplasmic extensions and characterised by a discontinuous basal lamina. Compared to SMC, myofibroblasts have a larger Golgi apparatus and abundant rough endoplasmic reticulum, both indicating their synthetic activity (Eyden 2005). The microfilaments are arranged in bundles (stress fibres). Microfilaments form ‘dense bodies’ where they accumulate and insert in plasmalemmal attachment plaques. The nucleus is mostly indented. Immunohistochemical studies have shown that myofibroblasts feature heterogeneous cytoskeletal phenotypes. Hence various types of myofibroblasts according to their content of specific cytoskeletal filaments such as alpha-smooth muscle actin, desmin, myosin and vimentin have been described (JostarndtFögen et al. 1998; Schürch et al. 1998). In contrast, SMC are usually characterised as smooth spindle-shaped cells with numerous membrane caveolae, which are enveloped by a continuous basal lamina. Their cytoplasm contains uniformly distributed microfilament bundles that run in parallel to the long axis of the cells. The nucleus is elongated with blunt ends (Zhang et al. 1997; Schürch et al. 1998; Eyden et al. 1999). However, definition of differentiated SMC is difficult, since variations in marker gene expression depend upon the functional state of the individual cell, as described e.g. for contractile cells of the airways or for vascular SMC (JostarndtFögen et al. 1998; Tang 2008). Precise data on the nature of contractile cells surrounding the tubular system of canine testis and epididymis are not available; the present study was performed to assess the distribution of these cells and to classify them with respect to their possible physiological function. Materials and Methods Samples and preparation of histological sections Testicles including the epididymis of six young adult dogs (Canis lupus familiaris) were obtained after routine orchiectomy requested by the dog owners. The dogs did not suffer from any known impairment of spermatogenesis. Three tissue blocks of a volume of approximately 0.5  2  2 cm were taken from each of these 12 testicles, so that all parts of the tubular system within the organs could be examined. Tissue samples were fixed in buffered formalin according to Lillie, in Methacarn or in Bouin’s solution (Romeis 1989), dehydrated and embedded in paraffin. Sections were cut at 3–5 μm thickness and routinely stained with Mayer’s haematoxylin and eosin. Immunohistochemistry single staining The presence of the cytoskeletal filaments smooth muscle actin, desmin, smooth muscle myosin and vimentin was studied immunohistochemically in order to detect and to classify contractile tissue elements. One section per tissue block and antibody (i.e., 36 sections per antibody) was mounted on poly-L-lysine coated slides, deparaffinised and rehydrated. Endogenous peroxidase activity was blocked with 0.6% H2O2 in methanol, nonspecific binding activity with normal goat serum (DakoCytomation, Glostrup, Denmark) in phosphate-buffered salt solution (PBS) at room temperature. Antigen retrieval by digestion with 0.5 mg protease from Streptomyces griseus (Sigma, Vienna, Austria)/ml PBS for 15 min at room temperature was required for detection of desmin, microwave pre-treatment (2 × 5 min in 0.01 M citrate buffer, pH 6.0) for the detection of vimentin and myosin. Sections were incubated overnight with primary antibodies (manufacturer and dilution, see Table 1) at 4 °C. Products of the immunoreaction of anti-desmin, anti-myosin and anti-vimentin were detected using the anti-mouse PowerVision Kit (Immunovision Technologies, Daly City, CA, USA). Binding of anti-smooth muscle actin was 4