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From the * Department of Anatomy and Cell Biology,
McGill University, Montréal, Québec, Canada; and the
Division of Urology, Department of Surgery,
Mount Sinai Hospital, Toronto, Ontario, Canada.
| Correspondence to: Dr Louis Hermo, Department of Anatomy and Cell Biology, McGill University, 3640 University St, Montréal, Québec, Canada H3A 2B2 (e-mail: lhermo{at}med.mcgill.ca). |
| Received for publication January 25, 2002; accepted for publication April 23, 2002. |
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Abstract |
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Key words: Electron microscopy, principal cells, apical blebs, apocrine secretion
Studies on various animal species have demonstrated that principal cells of the VD possess the machinery for endocytosis and merocrine secretion. Electron-dense tracers injected into the lumen of the rat VD were found in coated pits and vesicles, endosomes, multivesicular bodies, and lysosomes in a sequential and time-dependent manner, which, together with the presence of endocytic receptors on the principal cell surface, suggest that these cells are involved in the endocytosis of substances from the lumen (Friend and Farquhar, 1967; Hermo and de Melo, 1987; Andonian and Hermo, 1999c). Immunocytochemical and radioautographic studies, in addition to the morphological presence of numerous rER cisternae and a well-developed Golgi apparatus, have indicated that principal cells synthesize and secrete glycoproteins into the lumen via the classical merocrine manner involving secretory granules (Wenstrom and Hamilton, 1984; Burkett et al, 1987; Pailhoux et al, 1990; Andonian and Hermo, 1999c). Moreover, the rat VD synthesizes different subunits of glutathione S-transferases to protect spermatozoa while they are stored and/or transported through the VD (Andonian and Hermo, 1999b).
In addition to merocrine secretion, principal cells of the bovine, mouse, and rat VD appear to be involved in apocrine secretion whereby a portion of the apical cytoplasm of a principal cell protrudes between adjacent microvilli to eventually detach and be liberated into the lumen (Niemi, 1965; Agrawal and Vanha-Perttula, 1988; Manin et al, 1995; Renneberg et al, 1995; Andonian and Hermo, 1999a; Hermo et al, 2002), but this activity has yet to be demonstrated in the human VD.
Over 500 000 vasectomies are performed in United States every year, and 6% of these men eventually request reversal, despite the high cost and variable success rate (between 30% and 76%) (Jean-Francois et al, 1999; Potts et al, 1999). In rats, vasectomy results in dramatic changes to the epithelium of the epididymis (Flickinger et al, 1995; Flickinger and Howards, 2002); however, little attention has been paid to the VD itself. Vasectomy in dogs caused a dilatation of the lumen and a shortening of principal cells, along with a decreased number and size of their microvilli in the VD proximal (testicular) to the vasectomy site. No significant changes were noted in the VD distal (abdominal) to the vasectomy site (Wright and Hamidinia, 1983). In humans, as noted with the scanning electron microscope, the VD proximal (testicular) to the vasectomy site showed a reduction in thickness of the epithelium, with the majority of cells demonstrating stubby, apparently atrophic microvilli, but little change in the content of cytoplasmic organelles (Kiviat et al, 1978). However, the effects of vasectomy in humans under transmission electron microscope have not been studied in any great detail.
The purpose of the present study was thus twofold: 1) to examine the ultrastructural features of the epithelium of the scrotal VD from fertile men undergoing elective vasectomy and correlate these findings with studies on animal models, and 2) to examine the ultrastructural effects of vasectomy, obtained at the time of vasectomy reversal (vasovasostomy [VV] or vasoepididymostomy [VE]), on the scrotal VD at proximal (testicular) and distal (abdominal) regions relative to the initial site of vasectomy. These studies will provide clues as to the functions of the normal VD and the effects of vasectomy on the ultrastructural features of the VD.
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Materials and Methods |
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The no-scalpel vasectomy as a form of permanent male contraception was carried out (Li et al, 1991). Essentially, after the shaving, prepping, and draping of the scrotum, each VD was manipulated to a subcutaneous plane in the median raphe at the junction of upper one third and lower two thirds of the scrotum. Lidocaine (1%) was applied first as a small subdermal "wheal" and then infiltrated into the perivasal sheath. The scrotal skin was then punctured and dilated, and the scrotal VD was dissected out with vas dissection forceps. A 10- to 20-mm piece of VD was excised from both sides. Half of the specimen was sent to pathology, and the remainder was used for the present study. The cut ends of the VD were then cauterized and clipped with titanium clips and replaced into the scrotum, after which a small dressing was applied.
Patients undergoing microsurgical vasectomy reversal (VV or VE) had been vasectomized between 2 and 15 years earlier (median, 10 years). Such microsurgical procedures have been described previously (Silber, 1984). Pieces of the scrotal VD (10-20 mm in length) were obtained from proximal (testicular/epididymal) and distal (abdominal/prostatic) regions of the VD, 15-20 mm beyond the initial site of vasectomy, to avoid areas of fibrosis and scarring near the immediate site of vasectomy. We were not permitted to collect areas of the VD more distally. During vasectomy reversal procedures, seminal fluid from the testicular VD was examined under the light microscope to check for viable spermatozoa. Whenever motile spermatozoa could not be identified from the testicular VD, VE rather than VV was performed.
In all cases, VD tissue was kept on ice and was immediately fixed by immersion in 5% glutaraldehyde buffered in sodium cacodylate (0.1 M) containing 0.05% CaCl2 at pH 7.4. This fixative gave the best results. After 5 minutes of immersion, VD tissue was cut in cross section into small 1-mm3 pieces and placed in the same fixative overnight at 4°C. After a wash in buffer, the VD tissue was postfixed in potassium ferrocyanide—reduced osmium tetroxide for 1 hour to enhance the staining of membranes (Karnovsky, unpublished). Tissues were then rinsed several times in cacodylate buffer, dehydrated in ethanol and propylene oxide, and embedded in Epon 812. Thick sections (1 µm) were cut, stained with toluidine blue, and examined under the light microscope. Thin sections of selected areas were cut with a diamond knife, placed on copper grids, counterstained with uranyl acetate and lead citrate, and examined with a Philips 400 electron microscope (Philips, Eindhoven, The Netherlands).
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Results |
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In the electron microscope, principal cells showed cisternae of rER dispersed throughout the cytoplasm amid mitochondria and ribosomes/polysomes. The Golgi apparatus was conspicuous, as were endosomes, multivesicular bodies, and numerous lysosomes (Figure 2). The lysosomes presented several morphologies, including characteristic small- to medium-sized lysosomes with a homogeneous-dense matrix as well as numerous lipofuscin granules that showed an electron-dense homogeneous portion alongside a lipidic component (Figure 2). The nuclei of principal cells were highly lobulated (Figure 2). Junctional complexes connected adjacent principal cells to one another (Figure 2).
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The apical plasma membrane presented coated pits, vesicles, and microvilli extending into the lumen (Figure 2). Among the microvilli were apical blebs, projections of the apical principal cell cytoplasm into the lumen (Figures 2 and 3). Apical blebs had a consistent appearance among the principal cells of all fertile men examined. Unlike the apical cytoplasm of principal cells, which contained numerous rough cisternae of the rER, the Golgi apparatus, lysosomes, lipofuscin granules, and mitochondria, apical blebs still attached to the principal cell apex contained solely numerous ribosomes/polysomes and variable amounts of rER cisternae (Figures 2 and 3). The plasma membrane delimiting apical blebs did not show coated pits and microvilli (Figures 2 and 3).
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While some apical blebs showed continuity with the apical principal cell surface via a thin or broad base of attachment (Figures 2 and 3), others were far removed and did not appear to be connected to it (Figure 2). In the lumen, apical blebs took on various shapes and sizes. While all detached blebs contained numerous ribosomes and sparse amounts of rER cisternae, some also showed large cavities in their interior enveloped by large, dilated, irregularly shaped membranous profiles (Figure 2). Similar membranous profiles were readily found loosely distributed in the lumen (Figure 2). Several attached apical blebs extended far into the lumen and showed a more narrow proximal and bulbous distal portion (Figure 3). In such blebs, a segregation of their contents was noted, with the latter containing mainly ribosomes, while the former contained numerous rER cisternae. Points of constriction in such blebs between the proximal and distal portions suggested that such areas might represent the sites of detachment of blebs (Figure 3). In fact, some detached blebs had a slender process that closely approximated that from another apical bleb still connected to the principal cell surface, suggesting a recent separation of the bleb from its parent stalk (Figure 2).
Scrotal VD From Vasectomized Men Undergoing Vasectomy Reversal
In the light microscope, the VD proximal (testicular) to the site of
vasectomy showed a large dilated lumen and a slight reduction in size of the
epithelium (Figure 4, inset)
compared to normal fertile patients. Principal cells showed a paucity of
microvilli and apical blebs and numerous dense supranuclear granules
(Figure 4, inset). In the
electron microscope, such granules represented lysosomes showing an irregular
outline and moderately dense homogeneous matrix in which were embedded
electron-dense plaques, often with a rod-shaped appearance
(Figure 4). Microvilli were
sparse and stubby, and apical blebs were not frequent; those present contained
rER cisternae (Figure 4). The
nuclei of principal cells were irregular in shape and highly lobulated, with
thin strands connecting the various lobes
(Figure 4).
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In contrast, the VD distal (abdominal) to the site of vasectomy demonstrated dramatic changes. As seen in the light microscope, the epithelium was dramatically reduced in size, and a lumen was virtually nonexistent (Figure 5a and b). It is important to note that sections of the abdominal VD further removed from the site of vasectomy had a larger lumen, making vasectomy reversal possible. However, the epithelium and lumen were in no way comparable to that noted in fertile males. At times, numerous blood vessels appeared next to the diminished epithelium (Figure 5b), and at higher magnification, neutrophils abounded in the lamina propria adjacent to them as well as in surrounding blood vessels (Figure 5b, inset).
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In the electron microscope, the epithelium consisted of flattened principal cells bordering a thin inconspicuous lumen (Figure 5c). Principal cells extended from the basement membrane to the lumen but were so dramatically reduced in size that they lacked all of the structural features seen in principal cells of the scrotal VD from fertile men (Figure 5c). Absent were microvilli, apical blebs and the numerous lysosomes, rER elements, lipofuscin granules, and conspicuous Golgi apparatus of normal principal cells, suggesting an appearance of undifferentiated cells (Figure 5c). In addition, their nuclei were fairly regular in appearance, unlike the highly lobulated shape seen in normal cells (Figure 5c). Basal cells were not readily distinguishable, although portions of cells residing solely toward the base of the epithelium were noted (Figure 5c). Junctional complexes were present between adjacent principal cells (Figure 5c). The sample size of 7 patients was too small to characterize differences among patients at similar time intervals from the time of vasectomy to vasectomy reversal. However, all of the features described above were consistent findings among the 7 patients examined.
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Discussion |
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Apical blebs were noted in the present study to emanate from the apical principal cell surface, with some appearing to float freely in the lumen. Previous scanning electron microscopic studies of human VD had shown smooth protrusions or cytoplasmic extrusions of varying size and shape (4 µm in length and 0.8-2.5 µm in width) among the microvilli of principal cells (Brueschke et al, 1974; Kiviat et al, 1978). These spherical objects, which were non-nucleated, acidophilic, and associated with spermatozoa, were mistaken for foreign cells (Brueschke et al, 1974) and may represent the apical blebs noted in the present study.
Apical blebs have been reported in the epithelia of various ducts of the male reproductive tract of the rat, mouse, dog, monkey, and bull (see the reviews of Aumuller et al, 1999; Hermo et al, 2002; Hess, 2002). In humans, they have been reported in the epididymis, seminal vesicle, coagulating gland, and prostate (Rajalakshmi et al, 1993; Aumuller et al, 1999). However, this is the first transmission electron microscopic study examining their appearance and contents in the human VD.
Although once considered artifacts of fixation, apical blebs have been reported under the best perfusion fixation conditions. The fact that apical blebs are noted as distinct morphological features of principal cells from the middle and distal regions of the rat VD despite a similar perfusion fixation for the entire VD makes them less likely to be artifacts of fixation (Andonian and Hermo, 1999a). While it is generally agreed upon that perfusion fixation better preserves the ultrastructural features of tissues when compared to immersion fixation, perfusion fixation is not possible in human studies. Therefore, immersion fixation with 5% glutaraldehyde rather than the standard 2.5% glutaraldehyde was used to cross-link proteins and maintain structural integrity of organelles and cells. To further increase delivery of fixative to the epithelium, the pieces of VD were further cut into 1-mm3 pieces 5 minutes after immersion. The results obtained show cell-to-cell integrity as judged by intact junctional complexes and the absence of dilated organelles. Thus, apical blebs as seen in the present study are not likely to represent artifacts of fixation of human VD tissue.
Another criticism that apical blebs enjoy is that they may appear free-floating because the microscope plane of section is, by chance, missing the stalk that connects them to the parent cell. While we cannot exclude this possibility, which may be the case with some apical blebs, the fact that many blebs are deep in the lumen would suggest otherwise. Nevertheless, serial sections may be warranted in future studies.
Apical blebs contain solely numerous ribosomes and few ER cisternae, despite being in direct continuity with the apical cytoplasm of principal cells, which suggests a segregation of organelles between the 2 regions. In addition, some blebs appeared to detach as a distal bulbous portion from an extended stalk of cytoplasm still connected to the parent cell. While the mechanisms of segregation and separation are unknown, it has been hypothesized that apical plasma membrane proteins and the apical cytoskeleton participate in the formation of apical blebs and in the exclusion of apical organelles such as endosomes, lysosomes, mitochondria, and Golgi apparatus from blebs. Furthermore, cytoskeletal proteins have been implicated in the retraction of the connecting stalk from the distal terminal portion of the apical bleb (Aumuller et al, 1999).
In the present study, apical blebs lacked microvilli and coated pits, unlike the rest of the apical plasma membrane of principal cells to which they connected, suggesting a difference between these 2 plasma membranes. The contents of blebs show mainly ribosomes and few rER cisternae. Similar characteristics have been described for blebs of epithelial cells of various other male reproductive glands, suggesting that these cells may be involved in apocrine secretion (Aumuller et al, 1999; Hermo et al, 2002), and this may also be the case for the human VD.
Recently, a protein was cloned from apical blebs of the mouse VD and found to lack the hydrophobic signal sequence needed for transport to the ER. It was hypothesized that this protein was synthesized on free ribosomes in apical blebs and gained access to the lumen via apocrine secretion, where it coated the surface of sperm (Taragnat et al, 1990; Manin et al, 1995). In the present study, a major component of apical blebs of the human VD were free ribosomes. A similar function for ribosomes of apical blebs, therefore, could be hypothesized in the case of the human VD, and apocrine secretion may be the manner by which proteins would access the lumen.
In the present study, some detached apical blebs contained large dilated membranous profiles enveloping large empty-looking cavities. Similar structures were also found in large numbers loosely distributed in the lumen of the duct. While we cannot exclude the possibility that such membranous profiles are artifacts of fixation, similar structures were observed in the lumen of the rat VD, which was fixed by perfusion (Andonian and Hermo, 1999a). Hence, it is hypothesized that these membranous profiles may appear as the result of the eventual breakdown of blebs in the lumen of the VD. Their functional significance has yet to be determined. Future studies are aimed at isolating, purifying, and identifying proteins from apical blebs to confirm their functional roles.
At the light microscopic level, the epithelium of VD proximal (testicular) to the site of vasectomy showed a diminished epithelium, with shorter microvilli and numerous lysosomes. Somewhat similar findings have been reported in the human, monkey, and dog (Alexander, 1972; Flickinger, 1973; Kiviat et al, 1978; Kothari and Gupta, 1978; Wright and Hamidinia, 1983). We have further shown that these lysosomes contain electron-dense plaques that are not present in principal cells of fertile men, suggesting differences in the types of substances endocytosed as a result of obstruction secondary to vasectomy and an increased concentration of substances now present at this site, which normally would have moved downstream.
The scrotal VD distal (abdominal) to the site of vasectomy (15-20 mm beyond the site of vasectomy) was markedly different from that of the proximal region, as the lumen of the duct was virtually absent, and the epithelium was dramatically reduced in size to a layer of flattened cells. The latter also showed a paucity of organelles, and microvilli and apical blebs were conspicuously absent. An examination of sections of the distal (abdominal) VD collected revealed that areas more distal along these pieces showed a slightly larger lumen, allowing for successful anastomosis, although in no way was it comparable to that of the fertile men examined in this study. Whether or not the lumen is normal more distally along the VD could not be assessed, as such areas were not permitted to be collected in this study.
The synthesis and secretion of proteins by the VD have been demonstrated to be androgen-dependent (Pailhoux et al, 1990). However, no significant changes in systemic levels of androgens have been reported in vasectomized males (Johnsonbaugh et al, 1975). Thus, we hypothesize that one of the factors necessary for the maintenance of the integrity of the epithelium is the presence of luminal seminal fluids derived from areas further upstream. Their absence may lead to an undifferentiated state for the epithelial cells. The requirement of a normal patent lumen and its constituents being essential for epithelial integrity was also noted in the case of the efferent ducts, which after 14 days of ligation, showed major morphological changes to its epithelial cells, resulting in an undifferentiated state (Hermo and Morales, 1984).
The fibrosis surrounding the distal VD epithelium may be due to the initial vasectomy, even though the more grossly fibrosed areas were avoided during VV. The tissues obtained from vasectomized patients at the distal (abdominal) site also showed acute inflammation, as reflected by neutrophils within and outside numerous blood vessels in the lamina propria. This could be attributed to surgical trauma during VV/VE or ongoing complications resulting from vasectomy, as noted for the epididymis (Flickinger et al, 1995; Flickinger and Howards 2002). However, it should be pointed out that the epithelial cells did not show any signs of necrosis, degeneration, or apoptosis, as would be shown by abnormal chromatin patterns in the nucleus, indicating that the flattening of these cells was not due to these conditions. Thus, the findings in the distal VD appear to be the result of the undifferentiation of the epithelium as a consequence of vasectomy. These data on the distal and proximal VD may explain the low pregnancy rate after VV despite patent microsurgical anastomosis (Belker et al, 1991). In fact, vasectomy has also been shown to adversely affect the expression of various proteins secreted by the human and rat epididymis, which was not corrected by VV (Guillemette et al, 1999; Turner et al, 2000).
In the human VD, Kiviat et al (1978) did not note any dramatic effects of vasectomy on the distal (abdominal) VD. The difference between the 2 studies may be attributed to the shorter interval between vasectomy and reversal (4-8 years) used in their study, compared to that of the present study with a median of 10 years. These investigators may also have examined pieces of the distal VD from areas more remote from the actual site of vasectomy, which were not collected in our present study.
In another study on humans, in which the VD was ligated for only 2 weeks, Kothari and Gupta (1978) noted that the distal and proximal segments appeared to be essentially similar and normal. It could be argued that the interval of obstruction was again too short to notice any gross morphological changes. Similarly, studies on the dog VD showed that the distal (abdominal) VD remained unchanged (Wright and Hamidinia, 1983). Again, the interval of vasectomy was short, being only between 2 and 7 months in duration.
In summary, principal cells of scrotal VD from fertile men undergoing vasectomy showed apical blebs, suggesting that they are involved in apocrine secretion. In men undergoing vasectomy reversal, VD proximal (testicular) to the vasectomy site showed principal cells with reduced height, apical microvilli, and blebs but contained numerous lysosomes with electron-dense plaques, suggesting their active role in endocytosis. In contrast, the lumen of the VD distal (abdominal) to the vasectomy site was virtually abolished, with the epithelium being represented as a flattened layer of cells showing a paucity of organelles and no apical blebs or microvilli, suggesting that these cells become undifferentiated in the absence of seminal fluids and their constituents.
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Acknowledgments |
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Footnotes |
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References |
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