Mol. Hum. Reprod. Advance Access originally published online on February 25, 2005
Molecular Human Reproduction 2005 11(4):289-294; doi:10.1093/molehr/gah148
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Immunophenotypical characterization of contractile cells in caput epididymidis of men affected by congenital or post-inflammatory obstructive azoospermia
1Department of Internal Medicine, Andrology Unit, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy and 2Andrology Unit, S. Paolo General Hospital, Via A. Di Rudiní 8, Milano, Italy
3 To whom correspondence should be addressed at: Department of Internal Medicine, Andrology Unit, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy. E-mail: sandrof{at}univaq.it
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Abstract |
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Myoid cells of the human caput epididymidis are replaced by large cells with ultrastructural features of smooth muscle cells (SMC) in chronic obstruction of the male genital tract. To evaluate whether these cellular changes are associated with different functional phenotypes we analysed the immunohistochemical expression of myosin heavy chain isoforms and of extracellular matrix (EM) components in the human caput epididymidis contractile cells in normal and in obstructed epididymides. Normal caput epididymidis myoid cells expressed a scattered immunostaining for SM2, marker of differentiated contractile SMC, while no staining was detected for SMemb (the non-muscle-type myosin heavy chain isoform) and for its transcription factor BTEB2, markers of undifferentiated proliferating SMC. A faint immunoreaction (IR) for EM was observed in the peritubular wall of the normal caput. In the contractile wall of the obstructed caput epididymidis a strong IR was detected for all myosin heavy chain isoforms as well as for collagen type IV and for fibronectin, markers for a secretory function of SMC. These findings, unknown in other models of SMC pathophysiology, suggest that myoid cells resume the molecular machinery of both mature SMC and of differentiating/secretory cells in the chronic obstruction of the human caput. Contractile cells of the epididymal duct represent a unique model to study the plasticity of SMC.
Key words: collagen type IV/fibronectin/human epididymis/myoid cells/smooth muscle cells/smooth muscle heavy myosin chain isoforms
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Introduction |
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We have demonstrated that myoid cells of the human caput epididymidis are replaced in cases of chronic obstruction by differentiated Smooth muscle cells (SMC) (Pelliccione et al., 2004
), that under normal conditions are restricted to the cauda epididymidis and ductus deferens (Baumgarten et al., 1971; Francavilla et al., 1983). These SMC appearing in obstructed caput epididymidis show the co-existence of a contractile (large number of myofilaments coalescing into dense bodies) and synthetic (a developed Golgi complex and endoplasmic reticulum, scattered lipid inclusions and a thickened continuous basement membrane-like material) phenotype (Pelliccione et al., 2004). This suggests a phenotypic modulation of contractile cells in the adult epididymal duct.
SMC in the vascular system of adult animals retains the capability to acquire a broad spectrum of phenotypes (Owens, 1995), ranging from the contractile and fully differentiated to the proliferative/synthetic state, in response to different physiological or pathological stimuli (Chamley-Campbell et al., 1979; Thyberg et al., 1990). Smooth muscle myosin heavy chain isoforms are specific markers for SMC lineage (Nagai et al., 1988, 1989; Kuro-o et al., 1989; Aikawa et al., 1993) and their expression has been used to explore the different phenotypes of SMC in the development of vascular smooth muscles and in the atherosclerotic disease (Huszar, 1972; Robbins et al., 1986). SMC contains a developmentally regulated and cell-specific expression (Nagai et al., 1988, 1989; Aikawa et al., 1993; Miano et al., 1994) of muscle and non-muscle (NM) isoforms of myosin heavy chain (Rovner et al., 1986a; Nagai et al., 1989; Aikawa et al., 1993; White et al., 1993). SM2, a muscle isoform of myosin heavy chain, is expressed only after birth and is the best and most specific marker of fully differentiated SMC with a contractile phenotype (Kuro-o et al., 1989; Aikawa et al., 1993; Owens, 1995). SMemb, an embryonic NM isoform of myosin heavy chain expressed in fetal vessels (Rovner et al., 1986b; Kuro-o et al., 1991; Aikawa et al., 1993), is down-regulated during development (Rovner et al., 1986a), while it is up-regulated in immature or activated SMC during vascular injury (Gordon et al., 1990). Therefore SMemb is a marker for activated and immature SMC with a proliferative/synthetic phenotype (Kuro-o et al., 1991; Aikawa et al., 1993). The basic transcriptional element binding protein-2 (BTEB2) is involved in the transcriptional regulation of the SMemb gene during phenotypic modulation of SMC (Aikawa et al., 1993; Aikawa et al., 1998; Watanabe et al., 1999). The expression of BTEB2 is therefore an additional marker of activated SMC. The modulation of SMC to the synthetic state results in the production of extracellular matrix (EM) components that form thick basement membranes around the SMC (Clowes and Schwartz, 1985). An accumulation of collagen type IV and fibronectin, main components of basement membranes, has been demonstrated around activated SMC during vascular disease (Kurkinen et al., 1980; Murata et al., 1986; Shekonin et al., 1987).
In the present study, we examined changes in the contractile cell phenotype of human caput epididymis in normal conditions and in a congenital or acquired chronic obstruction. We report here an immunohistochemical (IH) study on human caput epididymis with the use of monoclonal antibodies against myosin heavy chain isoforms SM2 and SMemb, as well as for the transcriptional regulator of SMemb gene BTEB2. The expression of components of the EM was studied at the IH level with the use of monoclonal antibodies against collagen type IV and against fibronectin to analyse the secretory function of contractile cells. To our knowledge, this is the first report demonstrating diversity of contractile cell phenotypes in normal and abnormal human epididymides.
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Materials and methods |
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Epididymal specimens
Epididymal tissues were obtained from 15 patients. Five samples were normal epididymis from fertile men submitted to orchidectomy because of testicular cancer. Five specimens were from azoospermic men affected by congenital bilateral absence of vas deferens that also showed the absence of cauda and most of the corpus epididymidis. Five specimens were obtained from azoospermic men affected by bilateral congestive obstruction of the epididymis due to orchiepididymitis. Specimens in cases of obstructive azoospermia were obtained during epididymal sperm extraction carried out for assisted reproduction or during microsurgical epididymovasostomy. Particular care was devoted to collecting specimens of the head, proximal to obstruction level, as suggested by the gross appearance of dilated tubules containing a white creamy fluid. The samples obtained were fixed in 4% paraformaldehyde for 6 to 8 h and processed for routine paraffin embedding. A bilateral testis biopsy demonstrated a normal spermatogenesis in all 15 cases. The local ethics committee approved the study and the patients were requested to sign a consent form.
Specific antibodies
We employed monoclonal antibodies against human SM2, SMemb, BTEB2, with specificity already documented (Aikawa et al., 1993; Watanabe et al., 1999). Monoclonal antibodies against human Collagen Type IV (CP 56, Calbiochem, Darmstadt, Germany) and against Fibronectin (F 0916, Sigma, St Louis, MO) were used for EM components. Working dilutions were 1:400, 1:3000, 1:4000, 1:50 and 1:100, respectively for SM2, SMemb, BTEB2, Collagen type IV and Fibronectin.
Immunohistochemistry
After deparaffinization and rehydration, 3 µm-thick tissue sections were subjected to IH labelling according to the following protocol. Endogenous peroxidase activity was eliminated by pre-incubation with 0.3% (v/v) H2O2 in phosphate buffer saline (PBS) solution for 30 min at room temperature. Non-specific binding sites were blocked by incubation for 30 min at room temperature in normal goat serum in PBS. Sections were incubated overnight at 4 °C in PBS containing immunosera at dilutions mentioned above. After repeated washing in PBS, the immunoreaction (IR) was detected by an avidin–biotin peroxidase complex method using a biotinylated goat antimouse immunoglobulin G (Immunopure Ultra-Sensitive ABC Mouse immunoglobulin G Staining Kit; Pierce, Rockfort, IL, USA). Sections were then counterstained with hematoxylin solution for 4 min at room temperature, dehydrated and mounted. Controls were performed by the omission of primary antibodies (Figure 7). Mounted slides were examined in a Leica DM LB microscope (Leica icrosystem D-35578 Wetzler Germany).
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Myosin heavy chain isoforms and EM component expression in the normal proximal caput epididymidis
The epididymal caput showed a pseudostratified columnar epithelium formed by the principal and basal cells. Four to five overlapping regular circular layers of flat myoid cells formed a thin contractile (Figures 1, 2). This was well demarcated from the loose interstitial space and thin blood vessels were observed along their periphery. Immunohistochemistry for myosin heavy chain isoforms showed a faint and scattered immunoreaction (IR) for SM2 restricted to the cells, which constituted the outer layer of peritubular contractile wall, while no IR was present in the inner layer of the contractile or in the epithelial compartment (Figures 1, 2). NM myosin heavy chain SMemb expression was present in the brush-border of principal cells and along the epithelial basal compartment (Figure 3). Immunostaining for Smemb (Figure 3) and for its transcription gene regulator BTEB2 (Figure 4) was not present in the peritubular wall. No immunostaining for collagen type IV (Figure 5) and for fibronectin (Figure 6) was observed in the peritubular wall of patents' caput epididymidis.
Myosin heavy chain isoform and EM component expression in the caput epididymidis of men affected by chronic obstruction
The tubular lumen of the caput epididymidis was dilated and the contractile wall was strongly thickened in the caput epididymidis of men affected by chronic obstruction, as shown previously (Pelliccione et al., 2004). A strong expression for SM2 was equally observed in the inner and outer layers of peritubular cells (Figure 8). SMemb expression disappeared in the brush-border of principal cells while it was strongly expressed in the contractile wall (Figure 9) that also showed an immunostaining for BTEB2 (Figure 10). A strong IR for collagen type IV was observed in the contractile wall of obstructed caput epididymidis (Figure 11) and a continuous IR for this EM component regularly surrounded the contractile cells of the peritubular wall (Figure 12). A strong IR for fibronectin was also observed in the peritubular wall (Figure 13). No difference was observed in the expression of myosin heavy chain isoforms and EM components in the congenital and acquired epididymidis obstruction.
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Discussion |
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The present study demonstrates for the first time a phenotypic modulation of contractile cells of the human caput epididymidis in obstructive azoospermia, as determined through SM myosin heavy chain isoform expression.
During vasculogenesis in developing organisms, and during remodelling following vascular disease in adult organisms (Schwartz et al., 1986) SMC proliferates and shows EM synthesis (Kurkinen et al., 1980; Clowes and Schwartz, 1985; Murata et al., 1986; Shekonin et al., 1987) and partially lose the contractile phenotype typical of adult SMC (for review, see Owens, 1995). The modulation of SMC under physiological and pathological conditions is demonstrated by the different expression of myosin heavy chain isoforms (Nagai et al., 1988; Kuro-o et al., 1989; Nagai et al., 1989; Aikawa et al., 1993). In human vascular disease, differentiated SMC progressively reacquires a synthetic/proliferating phenotype. Indeed at the IH level, expression of SM2, a specific marker of fully differentiated contractile SMC (Kuro-o et al., 1989; Aikawa et al., 1993), is reduced (Kuro-o et al., 1991; Okamoto et al., 1992, 1996), while the expression of NM myosin heavy chain SMemb, a marker of undifferentiated proliferating vascular SMC during fetal life (Rovner et al., 1986b; Kuro-o et al., 1991; Aikawa et al., 1993), is increased (Kuro-o et al., 1991; Okamoto et al., 1992, 1996). These observations highlight the extreme plasticity of SMC that has the capacity to adapt their phenotypic state in response to physiological or pathological stimuli. Therefore, even in mature animals these cells can acquire a broad spectrum of phenotypes ranging from a contractile to a synthetic state (Chamley-Campbell et al., 1979; Thyberg et al., 1990).
The present study, by exploring for the first time the expression of SM myosin heavy chain in the human epididymis, showed that the normal adult caput epididymidis does not express NM myosin heavy chain SMemb and few peritubular cells express myosin heavy chain SM2. This suggests that myoid cells are quiescent, non-proliferating/non-synthetic and poorly differentiated contractile cells. The IH findings corroborate the ultrastructure of myoid cells. Indeed these cells retain only the elongated thin cytoplasm with a poorly developed synthetic apparatus coupled with the presence of thin bundles of contractile filaments (Pelliccione et al., 2004). The phenotypic modulation of contractile cells in the caput epididymidis when the patency of the duct is chronically lost due to obstruction of distal duct segments, suggests that myoid cells are a component of the SMC lineage. Indeed the ultrastructure of fully differentiated SMC that appears in the obstructed caput (Pelliccione et al., 2004) is associated with a strong expression of myosin heavy chain SM2, suggesting the development of the contractile phenotype typical of mature vascular SMC. Nevertheless, the same cells rescue a proliferating/synthetic phenotype judged by the expression of NM myosin heavy chain SMemb and of the SMemb transcription gene regulator BTEB2. The increased expression of the components of extracellular components such as fibronectin and collagen type IV, is in line with the rescue of a secretory phenotype of the SMC in the obstructed caput epididymidis. The production of EM components by the SMC during the synthetic state is known (Clowes and Schwartz, 1985), and an accumulation of collagen type IV and fibronectin has been demonstrated around activated SMC during vascular disease (Kurkinen et al., 1980; Murata et al., 1986; Shekonin et al., 1987).
Taking these observations together suggests that the lost patency of the epididymal duct triggers or is associated with a phenotypic modulation of contractile cells that resume a differentiation programme to fully mature SMC associated with a rescued secretory/proliferative state. Especially intriguing is the expression of NM myosin heavy chain SMemb and BTEB2 in the obstructed epididymal wall. SMemb is strongly expressed in adult coronary arteries during restenosis following percutaneous transluminal coronary angioplasty (Simons et al., 1993; Aikawa et al., 1997) and in proliferating mesangial cells in various types of glomerular disease (Leclerc et al., 1992), suggesting that SMemb may play a role in cell activation and/or cell division (Aikawa et al., 1997). The expression of SMemb suggests that in the caput epididymidis wall, in case of distal obstruction, the resumed differentiation programme is associated with a rescued proliferation of quiescent contractile cells. The contemporary expression of SMemb transcription gene regulator BTEB2 suggests an up-regulation of SMemb gene and is in line with the increased expression of SMemb mRNA shown in activated SMC of restenotic coronary arteries in adult men (Leclerc et al., 1992; Simons et al., 1993).
A key question is what controls the phenotypic changes of epididymal contractile cells when the patency of the duct is lost. Numerous studies in vivo and in vitro have demonstrated a modulation of myosin heavy chain gene expression in vascular SMC (Owens, 1995), but the molecular mechanisms underlying the different expression of myosin heavy chain genes are uncertain. There is a strong evidence that mechanical factors (shear stress and tangential wall stress) may play a key role in SMC differentiation during vascular development (Girard, 1973; Hu and Clark, 1989; Sterpetti et al., 1993); indeed the expression of many SMC differentiation markers of chicken embryos is accelerated with the establishment of blood flow and when blood pressure increases in developing vessels (Hu and Clark, 1989). Therefore, it is likely that the increase of mechanical forces acting on the chronically obstructed contractile epididymal wall, in addition to unknown local factors, triggers or is associated with a phenotypic modulation of contractile cells.
In conclusion the modifications of contractile cells of the caput epididymidis in the case of chronic obstruction (Pelliccione et al., 2004) are associated with a modulated expression of differentiated myosin heavy chain isoforms, indicating the activation of a differentiation programme associated with a proliferating/secretory programme resulting in the switch from quiescent poorly differentiated myoid cells to fully differentiated SMC with a coexistent proliferating/secretory phenotype. This relies on the remarkable plasticity of the SMC lineage, a component of which is the myoid cell that assures a continuous modulation of its function. It is expected that by microsurgical restoration of the duct patency, the SMC of the caput switch progressively and with time to less differentiated, quiescent and autocontractile cells able to ensure a continuous peristaltic progression of sperm throughout the genital duct. The reported long time laps of 6–12 months between the microsurgical repair of epididymidis obstruction and the reappearance of ejaculated sperm (Matthews et al., 1995) might be related to the adaptative changes of the contractile cells in the epididymidis duct.
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Acknowledgements |
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This project was supported by a grant from MIUR, COFIN 2003, Italy. The authors thank Prof. Jianglin Fan from the Department of Pathology, Institute of Basic Medical Sciences, University of Tsukuba, Japan, who kindly gifted monoclonal antibodies directed against human SM2, Smemb and BTEB2.
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Submitted on November 10, 2004; accepted on December 30, 2004.
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