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From the * Department of Anatomy and Cell Biology,
McGill University, Montreal, Canada;
INRS-Institut Armand Frappier,
Université du Québec, Pointe-Claire, Canada;
Département de Stomatologie,
Université de Montréal, Montreal, Canada; and
Department of Cell and Molecular Biology,
Feinberg School of Medicine, Northwestern University, Chicago, Illinois.
| Correspondence to: Dr Louis Hermo, Department of Anatomy and Cell Biology, McGill University, 3640 University St, Montreal, Quebec, H3A 2B2 (e-mail: louis.hermo{at}mcgill.ca). |
| Received for publication February 1, 2007; accepted for publication March 22, 2007. |
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Abstract |
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Key words: RT-PCR, immunocytochemistry, RCE cell line, orchidectomy, testicular extract
Principal cells, the most abundant epithelial cell type, have been implicated in diverse functions including the absorption and secretion of water, ions, solutes, proteins, and lipids (Hermo and Robaire, 2002; Turner, 2002; Breton, 2003). These activities allow for the formation of a specific luminal microenvironment that changes dramatically along the length of the epididymis, and it is in this highly specialized milieu that sperm undergo their maturational modifications (Hermo and Robaire, 2002; Turner, 2002; Breton, 2003). This microenvironment is maintained by the presence of a blood-epididymal barrier that is formed apically between adjacent epithelial cells and serves to maintain the necessary ionic composition and pH of the lumen (Cyr et al, 2002). In addition, tall microvilli extend from the apical plasma membranes of principal cells and form a uniform brush border with occasional branching. These microvilli, termed stereocilia, provide the cell surface with a large surface area potentially available to interact with molecules present in the lumen. Of particular note is the fact that microvilli contain a variety of membrane pumps, channels, and transporter systems and in this way maintain the proper volume as well as appropriate pH of both the principal cell and epididymal lumen (Wong et al, 2002; Turner, 2002; Hermo et al, 2002, 2005; Breton, 2003). While past studies have demonstrated that microvilli on principal cells are reduced in size after orchidectomy (Wahlqvist et al, 1996), the specific testicular factor(s) involved in their maintenance and the protein(s) involved has yet to be identified.
Microvilli are built around a common cytoskeletal element—the parallel actin bundle (Bartles, 2000). This bundle consists of tightly packed collections of actin filaments crosslinked by actin-bundling proteins, which serve to maintain many of the characteristics of microvilli. Three major classes of actin-bundling proteins have been detected in microvilli of vertebrate cells: villin, fimbrins/plastins, and espins (Bartles, 2000). Espins are actin-bundling proteins that come in multiple isoforms from a single gene and that differ markedly in size and their complement of ligand-binding sites (Sekerková et al, 2004, 2006a). The different espin isoforms or combinations of isoforms are expressed in different cell types and in complex spatiotemporal patterns during development (Sekerková et al, 2006a). In epithelial cells, they serve to elongate parallel actin bundles and thereby determine the steady-state length regulation and integrity of microvilli and stereocilia (Sekerková et al, 2006a).
The prototype espin was first identified as an approximately 110-kDa protein localized in the junctional plaque of the Sertoli cell ectoplasmic specialization of the testis, from which it derives its name: espin = ectoplasmic specialization + in (Bartles et al, 1996; Chen et al, 1999). To date there are 4 major espin isoform size classes ranging from about 110 to about 25 kDa and designated as espin 1–4 in order of decreasing size; splice variants are further specified alphabetically (Sekerková et al, 2004). Present in multiple actin-rich structures, espins are especially prominent in stereocilia of cochlear and vestibular hair cells and the microvilli of other sensory cells (Zheng et al, 2000; Sekerková et al, 2004, 2005). The stereocilia of principal cells of the human epididymis have been shown to contain an internal core of actin filament bundles crosslinked by fimbrin and associated with the membrane linker ezrin. Unlike brush border microvilli in the intestine and kidney, the microvilli of epididymal epithelial cells lack the bundling protein villin, but they also contain a different class of actin crosslinking protein, alpha actinin, in the stem portion of their long microvilli (Hofer and Drenckhahn, 1996).
In this study, we sought to explore how testicular factors regulate the growth and molecular composition of epididymal cell microvilli. These analyses were performed in vivo and using an immortalized rat caput epididymal (RCE) cell line, which has recently been developed by Dufresne et al (2005). We identify a specific espin isoform in the microvilli of epididymal principal cells and discover that its expression and microvillar dimensions are regulated by testicular factors.
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Materials and Methods |
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Experimental Protocols
In Vitro Regulation—
Immortalized RCE cells were grown on 6-well plates coated with collagen IV
in DMEM/HAM F12 medium (Sigma-Aldrich, Mississauga, Canada). Cells were
incubated at 32°C with 5% CO2. The cultured cells adhere to the
collagen and polarize with microvilli extending from 1 surface. The cells were
removed from the wells by trypsin digestion (0.05%). Procedures for culturing
these cells are described by Dufresne et al
(2005). Preparation of
testicular extract was as follows: adult rat testes were homogenized in
culture medium (2 vol/wt). Homogenates were centrifuged at maximum speed for
10 minutes at 4°C. The supernatant was collected and stored at
–86°C. The supernatant was filtered using a 0.22-µm filter prior
to use. Separate wells containing the RCE cells were cultured in the presence
of 0% (control), 0.05%, 0.5%, and 5% testicular extract (TE) for 48 hours. At
the end of the incubation period, the cells from 4 separate wells for each
experimental group were centrifuged at 1000 x g at room
temperature and the pellets fixed with 2.5% glutaraldehyde in 0.1 M sodium
cacodylate buffer (pH 7.4) for 24 hours at 4°C. Cells were washed
overnight in 0.1 M sodium cacodylate buffer, dehydrated in acetone, and
embedded in Epon. Ultrathin sections were cut with a diamond knife and mounted
on copper grids. Sections were stained with lead citrate and uranyl acetate
prior to examination by electron microscopy (JEOL-USA Inc, Peabody, Mass).
Electron micrographs of microvillar areas at 1 pole of the RCE cells exposed to different concentrations of testicular extract were taken at a raw magnification of 4300x from which prints were made at a final enlargement of 10 500x. The total boundary area occupied by microvilli projecting from the apical side of the cells was measured using a MOP-3 image analyzer (Carl Zeiss Canada Ltd, Toronto, Canada). The area occupied by microvilli projecting into this space was then outlined and summed, and the dead space between microvilli was computed as the difference between the total boundary area and the summed microvillar areas in the microscopic field. Microvillar areas from 70–85 different cells were measured in this fashion for each treatment group. Mean profile areas for the boundary, microvillar, and dead space in each treatment group and accompanying standard deviations and 95% confidence intervals were computed using Statistica software (version 7.1; Statsoft Inc, Tulsa, Okla). Initial analyses indicated that the data were not distributed normally, and log10 transformations were done prior to carrying out 1-way analysis of variance for multiple groups with unequal N, Student's t tests for independent samples (controls vs any one of the treatment groups), and power tests. Significance was established at P < .05.
In Vivo Regulation— Adult rats were anesthetized with an intraperitoneal injection of ketamine-xylazine (50:10 mg/kg). Four rats were sham operated as controls. Four other rats were bilaterally orchidectomized via an abdominal incision and killed 14 days later. Eight other orchidectomized rats were implanted with either an empty 2.5-cm capsule (4 rats) or 18.6-cm capsules (3 measuring 6.2 cm each) (4 rats) containing testosterone. Testosterone-filled polydimethylsiloxane capsules were prepared according to the method outlined by Stratton et al (1973) and have well-characterized steroid release rates (Brawer et al, 1983). These capsules mimic epididymal (18.6 cm) testosterone levels, which are 10 times greater than blood levels. Additional carrier rats were implanted with the testosterone implants prior to the start of the experiment to ensure that the implants functioned properly. These implants were removed from the carrier rats 3 days later, cleaned, and inserted subcutaneously on the backs of experimental animals at the time of orchidectomy. All of these rats were killed 14 days after surgery. At the completion of each experiment, the epididymides were fixed by retrograde perfusion with Bouin fixative and subsequently dissected and processed as described below for immunocytochemical analyses.
Efferent duct ligations were performed as follows. Adult rats (350–400 g) were anesthetized with an intraperitoneal injection of ketamine-xylazine (50:10 mg/kg). An abdominal incision was made through the peritoneum, and the testis and epididymis were gently retrieved. The efferent ducts were exposed, and the fat surrounding the ducts was displaced with tweezers. Using a black silk suture, the efferent ducts of each epididymis were ligated close to the rete testis leaving the vascularization intact. The testis and epididymis were then gently replaced in the scrotum and the incision sutured. Four sham operated rats were used as controls. Rats were sampled 14 days following surgery. At the end of the experiment, the epididymides were fixed by perfusion with Bouin fixative.
Identification of Espin mRNA in Adult Rat Epididymis— Four adult male rats were anesthetized with an intraperitoneal injection of ketamine-xylazine (50:10 mg/kg) and killed. Epididymides were removed and divided into 4 segments (initial segment [IS], caput [CT], corpus [CS], cauda [CA]); testes were used as positive controls. RNA was isolated from each segment using a commercial kit (Absolutely RNA RT-PCR Miniprep kit; Stratagene, Cedar Creek, Tex) according to the manufacturer's instructions. Espin-specific primers were designed according to the rat espin sequence. Reverse transcription was carried out using the reverse primer specific for the espin sequence (5'-TTC AAG ATG ACC TGT CGC TGC-3'). Specific forward and reverse espin primers (reverse: 5'-CTC GTC GTA TCC TAG TGT CCG-3'; forward: 5'-CCC TCA TCC CCA CAC TTG ATG AGC-3') were used for polymerase chain reaction (PCR). Espin was linearized at 94°C and amplified using 35 cycles of 94°C (30 seconds), 60°C (1 minute), and 68°C (1 minute). Resulting amplicons were separated on 1.5% agarose gels in Tris-Borate Ethylenediammine Tetraacetic Acid (90 mM Tris-Borate, 2 mM Ethylenediammine Tetraacetic Acid [EDTA]; TBE) buffer and stained with ethidium bromide (0.05 µg/mL). The intensity of bands was determined using a Bio-Rad Fluor Image analyzer (Bio-Rad Laboratories, Mississauga, Canada).
Immunocytochemical Localizations of Espin— Testes and epididymides from 6 adult male rats were fixed by perfusion through the abdominal aorta in a retrograde manner with either Bouin (n = 3) or St Marie fixative (n = 3). After 10 minutes of fixation, tissues were removed and placed for 24 hours in fresh fixative at room temperature and then placed in 70% alcohol for several days prior to dehydration and embedding in paraffin. Tissue sections of 5-µm thickness were cut and mounted on glass slides. Affinity-purified rabbit polyclonal anti-espin antibodies were utilized at a concentration of 10 µg/mL. These antibodies were purified and characterized as previously described (Bartles et al, 1996).
Tissue sections were deparaffinized in Histoclear (Fisher Scientific, Ottawa, Canada) and rehydrated by immersion in a series of graded ethanols. Immunocytochemistry was done using the DAKO Catalyzed Signal Amplification System (DAKO, Carpenteria, Calif) according to the manufacturer's instructions. Control sections were incubated with preimmune serum.
Immunofluorescence— Specimens fixed in St Marie fixative and embedded in paraffin were utilized for all immunofluorescent localizations done with adult in vivo epididymal tissues. Sections were rehydrated and blocked with phosphate buffered saline (PBS) + 5% bovine serum albumin before incubation with antiespin antibodies (diluted 1:200) for 90 minutes at 37°C. At the end of the incubation period, the sections were washed in PBS and subsequently incubated with a secondary antibody labeled with a Rhodamine Red-X tag (diluted 1:200; Jackson Immunoresearch, West Grove, Pa) for 30 minutes. Sections were washed in PBS and mounted in Vectashield containing DAPI (Vector Laboratories, Burlingame, Calif). Similar incubation procedures were carried out for the RCE cells fixed in ice-cold methanol. Control slides were incubated with preimmune serum.
Western Blot Analyses— Western blots were done according to the methods described by Sekerková et al. (2006b). Briefly, epididymides were homogenized in 20 vol (milliliters per gram of wet tissue) of 0.25 M sucrose, 3 mM imidazole-HCl, pH 7.4, containing 1 mM phenylmethylsulfonyl fluoride at 4°C using a 5-mL Potter–Elvehjem homogenizer (8 strokes, 3000 rpm) and immediately extracted at a concentration of about 40 mg of wet tissue per milliliter by heating at 100°C in sodium dodecyl sulfate (SDS) gel sample buffer for 3 minutes, with intermittent agitation on a vortex mixer. The samples were centrifuged at 16 000 x g in a microcentrifuge, and the resulting supernatants were resolved in SDS gels and analyzed on Western blots using the ECL system (GE Healthcare, Baie d'Urfé, Canada) with or without prior stripping according to the procedure recommended by the manufacturer.
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Immunofluorescent staining of the RCE cells with an anti-espin antibody revealed a polarized reaction over the apical region of these cells (Figure 3a), indicating that these cells express espin and that its localization resides over their microvilli. Immunofluorescence was also performed on the adult epididymis, where a reaction was observed over the microvilli of principal cells (Figure 3b). No reaction was noted when tissue sections were treated with preimmune serum (not shown).
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Discussion |
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Espin expression, however, was absent from the epithelial clear cells. The microvilli of these cells do not form a uniform brush border but rather display very long, irregularly shaped, branching structures. Clear cells are highly endocytic in nature (Hamilton, 1975; Robaire and Hermo, 1988; Hermo et al, 1994), and such microvilli may provide for more effective interactions of receptors on their surface with substances (proteins) needed to be removed from the lumen. The absence of espin from clear cells may provide their microvilli with more flexibility to perform such needed functions.
Parallel bundles of actin are important for cell shape and physiology, including their presence in microvilli, and are assembled through sequential action of multiple actin bundling proteins such as espin (Bartles, 2000). Parallel actin bundles are commonly associated with relatively long-lived specializations of the cell surface, such as microvilli, where they function in part as a scaffold to support evaginations or invaginations of the plasma membrane. Its proposed role with actin and presence in microvilli of principal cells would suggest that espin plays an important role in their microvilli. It has been suggested that espin plays a role in the outgrowth of actin filaments, forming the inner core of microvilli, and in this way is important for steady-state length regulation and integrity of microvilli (Loomis et al, 2003; Rzadzinska et al, 2005). In the epididymis, the presence of microvilli on the apical surface of principal cells allows for the proper transport of ions, solutes, and water across the epididymal epithelium, as these structures express various pumps, channels, and transporters and in this way maintain the luminal microenvironment in a manner that is essential for the maturation of spermatozoa (Hermo et al, 2000; Hermo and Robaire, 2002; Breton, 2003).
In the testis, earlier studies demonstrated that low-dose treatments with testosterone and estradiol led to the premature detachment of step 8 spermatids from Sertoli cells. It was concluded that adhesion molecules between these 2 distinct cell types were dependent on these hormones (O'Donnell et al, 2000). More recently, it was demonstrated that FSH regulates the formation of ectoplasmic specializations, whereas testosterone alone did not affect these junctions (Sluka et al, 2006). In the epididymis, it has been documented that following orchidectomy there is a decrease in epithelial cell height, a striking loss of apical microvilli from their surface, and lysosomal accumulation and vacuolation (Wahlqvist et al, 1996; Delongeas et al, 1987; Orgebin-Crist and Davies, 1974; Moore and Bedford, 1979). This led to the suggestion that factors emanating from the testis are important in the proper maintenance of epididymal structure and functions. It is now well documented that such factors could reach the epididymis via the circulation and epididymal lumen (Robaire and Hermo, 1988; Cornwall et al, 2002; Robaire et al, 2006). In the latter case, factors of testicular origin entering the epididymis via the lumen are referred to as lumicrine factors, and several have been identified as regulating specific protein expression in the epididymis (Hinton et al, 2000; Robaire et al, 2006).
In the present study, while efferent duct ligation lowered the expression of espin, it was not entirely abolished, suggesting that espin expression could in part be regulated by circulating levels of androgens or a lumicrine factor(s). However, the fact that orchidectomy resulted in the complete absence of a reaction for espin over microvilli of principal cells and that microvillar espin levels were restored to control levels by administration of high doses of testosterone suggested that espin is regulated by androgens. Because testosterone levels in the lumen of the epididymis are approximately 10 times higher than in the blood (Turner, 2002), the levels in ligated rats appear to be insufficient in maintaining normal expression levels of espin. Hence, regulation of espin and microvillar size by androgens indicates their importance in regulating processes implicated in creating a luminal environment that is necessary for sperm maturation. Past studies have shown that many epididymal proteins are activated or down-regulated after orchidectomy (Orgebin-Crist et al, 1975; Holland et al, 1992; Robaire and Viger, 1995; Ezer and Robaire, 2002, 2003; Robaire et al, 2006). The present data suggest that androgens regulate espin expression in the adult epididymis.
In the male reproductive tract, many proteins have been shown to be
regulated by estrogens in the efferent ducts and epididymis
(Hess et al, 2002). To this
end, we examined whether or not espin is regulated by estrogen. To access this
parameter, we utilized wild-type and 
ERKO mice in conjunction with LM
immunocytochemistry on a number of epididymal slides from such mouse models
that we have used in previous studies (Ruz
et al, 2006). Espin expression was intensely expressed on the
microvilli of principal cells of wild-type mice and remained so in ERKO
mice, suggesting that estrogen did not regulate espin expression in the
epididymis (unpublished data).
Previous studies have shown that androgens regulate the expression of proteins implicated in forming adherens and tight junctions that are necessary for the formation of an intact blood-epididymal barrier (Cyr et al, 2002). Likewise, several pumps, transporters, and channels present on microvilli have also been shown to be androgen dependent (Badran and Hermo, 2002; Pastor-Soler et al, 2002; Ruz et al, 2004). Thus, one of the critical roles of androgens is to regulate epididymal functions that control the composition of epididymal lumen and hence sperm maturation, and this may in part be due to structurally and functionally active microvilli.
In summary, in the present study microvillar size in the RCE cell line is dependent on testicular factors, and espin is expressed on 1 pole of these cells. In the adult rat epididymis, espin is localized to the microvilli of principal cells, where it is regulated by high levels of testosterone. It is suggested that microvillar size and integrity are dependent on espin and its regulation by androgens.
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Acknowledgments |
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Footnotes |
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