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From the * Andrology Unit, Department of Internal
Medicine, University of L'Aquila (I), and the
Andrology Unit, S. Paolo General Hospital,
Milano, Italy.
| Correspondence to: Prof Sandro Francavilla, Dipartimento di Medicina Interna, Università dell'Aquila, Via Vetoio, 67100 L'Aquila, Italy (e-mail: sandrof{at}univaq.it). |
| Received for publication October 24, 2003; accepted for publication January 9, 2004. |
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Abstract |
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Key words: Congenital absence of vas deferens, human caput epididymidis, smooth muscle cell
The transport and storage of spermatozoa depend on a peculiar organization of the epididymal tubular wall. This consists of a mantle of contractile cells that change in the manner in which they are organized from the proximal caput to the ductus deferens both in humans (Baumgarten et al, 1971) and in rodents (Francavilla et al, 1983). A thin sheet of multilayered, flat myoid cells form the contractile wall from the ductuli efferentes to the proximal corpus. Large cuboidal mature smooth muscle cells (SMCs) superimpose on myoid cells in the distal corpus and in the cauda. Myoid cells are completely replaced by a thick layer of large SMCs at the beginning of the vas deferens.
A spontaneous rhythmic contractility of myoid cells (Suvanto and Kormano, 1970) is responsible for the continuous peristaltic movements required to move the spermatozoa along the efferent tubules, caput, corpus, and proximal cauda epididymidis (Muratori and Contro, 1951; Risley, 1958). In contrast, the sporadic, brief, forceful nerve-mediated reflex contraction of SMCs guarantees the storage of spermatozoa and its rapid disposal at ejaculation in the distal cauda and ductus deferens (Cross, 1959; Baumgarten et al, 1971).
Although the normal human epididymis is fairly well understood both at the morphologic and functional levels, the abnormal epididymis that is responsible for human infertility still remains an elusive issue (de Kretser et al, 1998). Obstructive azoospermia is the only known natural model of epididymal infertility. This is mainly because of a congenital bilateral absence of vas deferens (CBAVD) or a congestive obstruction. CBAVD, which accounts for at least 6% of the cases of obstructive azoospermia and is responsible for 1%–2% of the cases of male infertility (Vohra and Morgentaler, 1997), is a condition inherited as an autosomal dominant disorder, and it is considered a mild form of cystic fibrosis (Dumur et al, 1990; Culard et al, 1994). CBAVD is present in 95% of the male patients with the classic form of cystic fibrosis, and mutations in the gene encoding the cystic fibrosis transmembrane regulator (CFTR) have been identified in 60%–70% of patients with CBAVD (Anguiano et al, 1992; Dörk et al, 1997). Congestive obstruction of the epididymis in the adult is the result of orchiepididymitis (Chan and Schlegel, 2002a,b).
Although fine changes in the epididymal epithelium have occasionally been described (Rajalakshmi et al, 1993), no information, to our knowledge, is available on changes of the contractile wall of the human epididymis in cases of obstructive azoospermia. The enigmatic observation that the time taken for spermatozoa to appear in the ejaculate is very high (ranging from 6 to 12 months) after the microsurgical repair of obstructive azoospermia (Matthews et al, 1995) prompted us to hypothesize that the obstructive condition is associated with structural changes in the contractile wall that result in an altered transport and storage function of spermatozoa when the patency is restored.
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Materials and Methods |
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Light and Electron Microscopy
Tissue samples were rapidly fixed by immersion in 2.5% glutaraldehyde in
cacodylate buffer, pH 7.2, for 2 hours at 4°C and were postfixed in an
unbuffered osmium tetraoxide solution (1%) at 4°C for 1.5 hours. The
samples were dehydrated in alcohol and embedded in Epon 812 (AGAR Scientific
Ltd, Milan, Italy). Thick sections for light microscopy were stained with
buffered toluidine blue (pH 8.0) and examined in a Leica DM LB microscope
(D-35578; Leica Microsystems, Wetzler Germany).
The thickness of the tubular wall of the proximal caput was measured in each of the 15 cases with the Leica imaging system QWin. Twenty random measurements were collected from cross-tubule sections in the same region of the caput from each case.
For ultrastructural analysis, silver-to-pale golden ultra-thin sections were stained with uranyl acetate and lead hydroxide (AGAR Scientific) and examined in a Philips M100 transmission electron microscope (Philips Electronics, Eindhoven, Holland).
Statistical Analysis
To analyze the thickness of the tubular contractile wall in the 3 groups of
specimens, mean values from each group were compared using a 1-way analysis of
variance for repeated measures with the PROC GLM procedure. A post hoc
comparison between pairs of groups was assessed by the 2-tailed unpaired
t test with a downward adjustment of the 
level to compensate
for multiple comparisons. To maintain the overall probability at a level of
.05 in the 3 independent comparisons, the value was divided by 3 to
obtain a comparison-wise = .017 (.05/3). Thus, each comparison was
significant at the .017 level. Data analysis was performed with
SAS/statistical software (Statistical Analysis Systems Institute Inc, Cary,
NC).
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Results |
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Light Microscopy
Normal Caput Epididymis—
The epididymal proper duct showed a pseudostratified columnar epithelium
formed by principal and basal cells. A thin contractile wall was arranged in
4–5 overlapping regular circular layers of flat cells and showed a
constant thickness throughout the head
(Figures 1 and 2). Peritubular
cells appeared remarkably long, with a spindle-shaped nucleus and a light
cytoplasm, and were separated from each other by narrow spaces containing an
indistinct material (Figure 2).
The contractile wall was well demarcated from the loose interstitial space,
and thin blood vessels were observed along their periphery
(Figure 2).
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Obstructed Caput Epididymis— Substantial changes were observed in obstructed cases compared to patent cases, while no differences were observed between congestive obstruction (Figures 3 and 4) and congenital obstruction (Figures 5 and 6). The tubular lumen was dilated, and the epithelial columnar principal cells in some cases showed a decreased height, whereas others did not show any variation in the cellular height compared to the patent epididymis (Figures 3 and 5). The most relevant changes were observed in the contractile wall and involved both architectural and cytologic differences compared to normal cases. The wall was strongly thickened and disarrayed. An abundant amorphous substance accumulated between poorly oriented peritubular large cells, which completely replaced the spindle-shaped cells that were observed in the patent cases (Figures 4 and 6). Moving from the inner to outer peritubular layers, the contractile wall was progressively disorganized, causing the boundary of the wall to be loosely demarcated from the intertubular tissue (Figure 5).
The quantitative image analysis showed a significantly increased thickness of the contractile wall in the 2 obstructed groups compared to the controls (mean ± SD: 62.98 ± 5.84 µ, 80.82 ± 7.72 µ vs 19.59 ± 2.23 µ, respectively, for congestive and congenital obstructions vs controls; P < .0001 for both obstructed groups vs controls). The mean wall thickness in congenital obstruction was higher than in congestive obstruction, although the difference was not significantly different (P = .053).
Electron Microscopy
Normal Caput Epididymis—
The peritubular contractile wall of normal human epididymis contained
multilayered, overlapping, flat myoid cells separated by narrow intercellular
clefts containing thin collagen bundles and an amorphous substance. Myoid
cells contained a pale cytoplasm and elongated nuclei oriented along the major
cellular axis (Figure 7). A
discontinuous electron-dense basement membrane–like substance surrounded
the cellular plasma membrane, and scattered micropinocytotic vesicles were
observed along the plasma membrane (Figure
8). The contractile nature of these cells was suggested by the
presence of packed bundles of thin filaments (5–6 nm in diameter)
converging to scattered dense bodies
(Figures 10 and 11). Isolated
cells with a pale cytoplasm (Figure
7) did not show myofilaments
(Figure 9). Their cytoplasm
contained a well-developed endoplasmic reticulum and a Golgi complex in a
perinuclear position (Figures 9 and
10), while micropinocytotic vesicles were observed on the plasma
membrane (Figure 9).
Fibroblasts were intermingled between contractile elements. No cytologic
differences of contractile cells were observed in the inner and outer regions
of the tubule wall.
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Obstructed Caput Epididymis— The same architectural and cytologic features were observed in the contractile wall in congestive and congenital obstructions. The most relevant finding was the switch of the contractile elements from the phenotype of a myoid cell to that of an SMC. Cytologic differences were observed between the inner and outer layers of the contractile wall.
Inner Tubular Layer— The flat, spindle-shaped myoid cells were partially replaced by large cells with a polymorphic nucleus, while wide intercellular clefts were filled with an amorphous substance and collagen bundles (Figure 12). The cells showed an irregular contour, while the cytoplasm contained a developed Golgi complex (Figure 13) and endoplasmic reticulum (Figure 14), numerous mitochondria, and scattered lipid inclusions (Figure 15). Contractile thin filaments were restricted to the cellular periphery and progressively extended to the wider cellular area moving to the outermost layer of contractile cells (Figures 15 and 17). Micropinocytotic vesicles were scattered along the plasma membrane in contractile cells (Figure 17), and a discontinuous basement membrane–like material surrounded the innermost cells (Figure 12), while it appeared continuous in the outermost cells (Figures 15 and 17). Cells with a pale cytoplasm were not observed.
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Outer Tubular Layer— Isolated large cells (Figure 18) interspersed among considerable amounts of collagen and an amorphous substance (Figure 19) were present in the outer tubular layer and showed the phenotype of mature SMCs. A thick basement membrane–like material continuously surrounded the cellular plasma membrane (Figure 20), and the cytoplasm was particularly rich in thin myofilaments converging to a large number of dense bodies (Figure 20). A developed endoplasmic reticulum winding around the mitochondria was also visible (Figure 21). Small glycogen pools were scattered among myofilament bundles (Figure 22), and the plasma membrane showed an irregular outline because of the presence of crowded micropinocytotic vesicles (Figure 23).
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Discussion |
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The regional morphologic and functional differences of the epididymis contractile wall and the changes associated with a long-lasting total obstruction such as those we have reported represent a unique natural model to explore the plasticity of the SMCs in the genital tract and its possible clinical relevance.
Under physiologic conditions, poorly differentiated contractile cells (myoid cells) ensure that sperm are transported continuously along the efferent tubules, caput, corpus, and proximal cauda epididymidis through a spontaneous rhythmic contractility (Muratori and Contro, 1951; Risley, 1958; Suvanto and Kormano, 1970). The phenotype of contractile elements shifts to well-differentiated SMCs in the distal cauda and vas deferens (Baumgarten et al, 1971; Francavilla et al, 1983). The sporadic nerve-mediated reflex contraction of these regions of the excurrent duct guarantees the storage of spermatozoa and its rapid disposal at ejaculation (Cross, 1959; Baumgarten et al, 1971).
Our study shows that when the patency of the epididymis tubule is lost, either because of a congenital absence of the distal epididymal segments or because of orchiepididymitis, a thickening of the peritubular contractile wall in the caput proximal to the obstruction is associated with an accumulation of collagen and ground substance and with a switch in the contractile elements from myoid cells to SMCs, a phenotype that is normally restricted to the cauda epididymidis and ductus deferens. In addition, SMCs in the obstructed cases showed ultrastructural features that are associated with a contractile activity (a large number of myofilaments coalescing into dense bodies) coexisting with those of a synthetic secretory activity (a developed Golgi complex and endoplasmic reticulum, scattered lipid inclusions, and a thickened continuous basement membrane–like material).
SMCs with a synthetic secretory phenotype were extensively described in a vascular system during arteriosclerosis (Stary, 1990), and the current opinion is that those cells that show an enhanced ability to synthesize extracellular matrix proteins are derived from fully contractile vascular SMCs (Clowes and Schwartz, 1985; Schwartz et al, 1985).
The origin of SMCs in the caput epididymis and the stimuli that trigger their differentiation are unknown. Myoid cells in the inner peritubular layer of the obstructed caput were only partially replaced by large cells, with an intermediate phenotype between the myoid cell and the SMC, while differentiated SMCs were the only cellular type present in the outer layer. This gradient of differentiation suggests that myoid elements gave rise to SMCs with coexisting contractile and secretory phenotypes, which resulted in the accumulation of extracellular matrix components.
A limitation to this hypothesis of a switch from myoid cells to SMCs is that it relies exclusively on ultrastructural findings. Indeed, cell markers able to specifically identify the 2 contractile cell types in the genital system are not yet identified. Among various markers of SMC differentiation proposed for vascular contractile cells, the differential expression of the heavy subunits of myosin seems to be a valuable tool for studying the structural modifications that these cells undergo during angiogenesis and in vascular disease (Owens, 1995; Sartore et al, 1997). Whether these and other differentiation/maturation markers of vascular SMCs can be applied to differentiate the phenotype of contractile cells in the human normal and obstructed epididymis is a matter of ongoing study.
The complex changes in the phenotype of the contractile cells that are observed in the obstructed epididymis suggest that modifications in local environmental cues normally required for the maintenance of their behavior play a pivotal role. The differentiation/maturation of contractile cells in the rat epididymis is temporarily coincident with the secretion of testicular fluid from the seminiferous tubules (Francavilla et al, 1987). This suggests that mechanical forces play a role in the differentiation of epididymal contractile cells, as is suggested for the vascular SMCs during angiogenesis (Hu and Clark, 1989).
The concept of mechanical forces as a mechanism that controls the differentiation of contractile cells in the epididymis may be applied to predict modifications that are associated with lumen obstruction. An increased wall stress, like that present in the epididymis tubule, proximal to the point of obstruction, has been shown to induce, by still unknown stimuli, an increased contractile mass in the vascular wall due to a hypertrophy of the SMCs (Owens et al, 1981). Therefore, the increased mechanical forces on the epididymal wall, proximal to the obstruction, may eventually activate the differentiation of myoid cells into SMCs.
Together, the findings reported in this study suggest that a chronic obstruction in the human epididymis is associated with a dramatic modification of the architecture and phenotype of the peritubular contractile cells in the caput, proximal to the obstruction. This relies on the remarkable plasticity of these contractile cells that, under the influence of different, not yet identified factors, undergo a continuous modulation of their functions.
An intriguing question is whether the adaptive mechanism of contractile cells in the obstructed caput epididymis contribute to the explanation of why, after microsurgical repair, 6–12 months might be required for spermatozoa to appear in the ejaculate (Matthews et al, 1995). This clinical finding suggests that the structural changes of the contractile wall associated with the obstructive condition result in an altered transport and storage of spermatozoa. These functions are eventually restored after the surgical repair of the obstruction, but the SMCs probably require some time to realign their phenotype and behavior before the reproductive system can be restored to normal functioning.
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Footnotes |
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