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From the Department of Anatomy and Cell Biology, McGill University, Montreal, Canada.
| Correspondence to: Dr Louis Hermo, Department of Anatomy and Cell Biology, McGill University, 3640 University St, Montreal, Canada H3A 2B2 (e-mail: louis.hermo{at}mcgill.ca). |
| Received for publication November 5, 2003; accepted for publication February 4, 2004. |
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Abstract |
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Key words: Water transport, Sertoli, basal, nonciliated, androgens
-helical domains
and has its own distinct pore to allow the bidirectional transport of water
(King and Agre, 1996;
Wintour, 1997;
Brown et al, 1998; Verkman and Mitra, 2000). To
date, 11 AQPs (0–10) have been found in different tissues. They have
been divided into 2 groups on the basis of their permeability properties: the
water-selective AQPs and the aquaglyceroporins, which, in addition, permeate
urea, glycerol, and other uncharged molecules
(Preston and Agre, 1991;
Deen and van Os, 1998;
Borgnia et al, 1999; Hatakeyama et al, 2001;
Sansom and Law, 2001). AQPs are expressed throughout the mammalian body and have been studied extensively (Verkman and Mitra, 2000; Nielsen et al, 2002; Schrier and Cadnapaphornchai, 2003). Many are tissue-, region-, and even cell-specific, and more than 1 AQP can be expressed on the same cell type (King and Agre, 1996; Echevarria and Ilundain, 1998; Verkman and Mitra, 2000; Nielsen et al, 2002). While hormones regulate some AQPs, others are constitutively expressed (Verkman and Mitra, 2000; Nielsen et al, 2002; Schrier and Cadnapaphornchai, 2003). Alteration in expression of AQPs has been shown to result in a variety of pathological states (King et al, 2000; Verkman and Mitra, 2000; Nielsen et al, 2002; Schrier and Cadnapaphornchai, 2003).
The transport of water in the male reproductive tract is essential for its various functions. In seminiferous tubules of the testis, water is secreted into the lumen by Sertoli cells in order to create the fluid environment essential for maintaining spermatogenesis and in serving as the vehicle to move sperm from the testis and through the efferent ducts into the epididymis (Setchell et al, 1969). In the efferent ducts, up to 90% of the testicular luminal fluid is reabsorbed, and fluid is constantly reabsorbed and secreted along the epididymis to concentrate the sperm so that they can have the proper environment to become fertile and motile (Ilio and Hess, 2002; Wong et al, 2002).
In the male reproductive tract, the distribution and regulation of several members of the AQP family have been studied in some detail (Brown et al, 1993; Andonian and Hermo, 1999; Nihei et al, 2001; Pastor-Soler et al, 2001; Badran and Hermo, 2002). In the rat, AQP-1 and AQP-9 have been localized to epithelial cells of the efferent ducts, and AQP-9 is expressed in principal cells of the epididymis in a region-specific manner (Fisher et al, 1998; Elkjaer et al, 2000; Pastor-Soler et al, 2001; Badran and Hermo, 2002). AQP-1 is also localized to the endothelial cells of vascular channels throughout the efferent ducts and epididymis (Badran and Hermo, 2002). In the testis, AQP-9 is localized to Leydig cells of the interstitial space, and while AQP-7 is expressed in germ cells, AQP-8 is expressed in Sertoli cells of the seminiferous epithelium (Ishibashi et al, 1997a; Nihei et al, 2001; Badran and Hermo, 2002). Various studies have shown that neither estrogen nor testosterone regulates expression of AQP-1 over the microvilli of the nonciliated cells and that expression of AQP-9 in principal cells of the epididymis is dependent on different factors in different epididymal regions (Zhou et al, 2001; Badran and Hermo, 2002).
Since many members of the AQP family are widely expressed in a given tissue such as the intestine and kidney, where they perform a variety of important functions, and considering the similar embryological derivations of regions of the male excurrent duct and the kidney, we undertook to examine the distribution of several other AQP family members in the different cell types of the efferent ducts and epididymis, as well as the testis. Despite localizations of AQP-1, AQP-2, and AQP-6 to AQP-9 in different regions of the male reproductive system (Hermo and Robaire, 2002), AQP-0, AQP-3, and AQP-10 have not as yet been studied in the male tract. Multiple expressions of AQPs in a given tissue may suggest diverse functions as well as the overall importance of AQPs in that tissue. In the testis, AQPs may play an indirect role in maintaining spermatogenesis, while in the efferent ducts and epididymis, they may provide the proper luminal environment for the transport and maturation of sperm.
In the present study, we examine the immunocytochemical localization of AQP-0, AQP-3, and AQP-10 in the different cell types of the testis, efferent ducts, and epididymis of normal adult rats. In addition, we examine the regulation of AQP-3 expressed in the epididymis in efferent duct ligated and orchidectomized rats with or without testosterone supplementation. The results demonstrate that a cell- and tissue-specific expression was noted for AQP-0, AQP-3, and AQP-10 in the control adult testis, efferent ducts, and epididymis, as well as differential regulating factors for the expression of AQP-3 in the epididymis.
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Materials and Methods |
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Light Microscope Immunocytochemistry
For control animals, the testes, efferent ducts, and epididymides of each
rat were fixed by perfusion with Bouin fixative via the abdominal aorta for 10
minutes. For experimental animals, only the epididymides were collected after
perfusion fixation. Following perfusion, the various tissues were removed, and
the epididymides were cut so that given sections would include all the major
regions of the epididymis (ie, the initial segment, intermediate zone, caput,
corpus, and cauda) (Hermo et al,
1991). The tissues were then immersed in Bouin fixative for 72
hours, after which they were dehydrated and embedded in paraffin.
Immunoperoxidase staining of sections was carried out according to the procedure of Oko and Clermont (1989). Polyclonal, affinity-purified anti-AQP 0, 3, and 10 antibodies were tried at different dilutions (1:50–1:200) in Tris-buffered saline, pH 7.4, with the 1:100 dilution showing the optimal reaction for the type of fixation and immunostaining method used. The anti–AQP-0 and AQP-10 antibodies were obtained from Alpha Diagnostics International (San Antonio, Tex). The anti–AQP-3 antibody was obtained from Santa Cruz Biotechnology Inc (Santa Cruz, Calif). The antibodies, well characterized and specific for their respective peptides, were purified from ascites fluid by protein G chromatography and raised against a 17–amino acid synthetic peptide for AQP-0 (Shiels and Bassnett, 1996), an 18–amino acid synthetic peptide for AQP-3 (Ishibashi et al, 1994), and a 17–amino acid synthetic peptide for AQP-10 (Hatakeyama et al, 2001), all within the carboxy termini of the proteins. They were supplied as solutions of 1 µg/µL in phosphate-buffered saline (PBS), pH 7.4, with 0.2% bovine serum albumin as a stabilizer. The antibody solutions also contained 15 mM sodium azide as a preservative.
Paraffin sections, 5 µm thick, were deparaffinized (Histoclear; Diamed Lab Supplies Inc, Mississauga, Canada) and hydrated in a series of graded ethanol solutions. During hydration, residual picric acid was neutralized in 70% ethanol containing 1% lithium carbonate, and endogenous peroxidase activity was abolished in 70% ethanol containing 1% (vol/vol) H2O2. Once hydrated, the tissue sections were washed in distilled water containing glycine to block free aldehyde groups.
After rinsing with tap water and PBS, sections were incubated in normal blocking serum (Vectastain Elite ABC kit, Vector K-6101; Vector Laboratories, Burlingame, Calif) for 30 minutes and then with the polyclonal anti-AQP antibodies, diluted to 1: 100 with PBS. Sections were then washed 3 times with PBS and incubated with biotinylated secondary antibody (ABC kit) for 30 minutes. After washing 3 times with PBS, sections were incubated with the ABC reagent for 30 minutes and finally washed again 3 times with PBS. Visualization of the stain was achieved by incubating sections (0.05% diaminobenzidine tetrahydrochloride; Bio FX Laboratories, Owings Mills, Md) until the desired staining intensity was achieved. Slides were rinsed with tap water for 5 minutes and then counterstained with 0.1% methylene blue. Passing them through a graded ethanol series dehydrated the tissues. Thereafter, the tissue sections were mounted on glass slides with Permount for observation. Negative controls were obtained by omission of the primary antibody or by use of normal rabbit serum.
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In addition, this pattern of staining was restricted to specific stages of the cycle. At the early stages (stages I–V), only about one fourth of the tubular circumference showed intensely reactive Sertoli cells (Figure 1a). At stages VI–VIII, intensely reactive Sertoli cells enveloped approximately one half of the tubular circumference (Figure 1b). At later stages (IX–XIV), a similar staining pattern was observed, but Sertoli cells at these stages were either weakly reactive or completely unreactive (Figure 1c). Thus, AQP-0 expression in the seminiferous epithelium was restricted to Sertoli cells and was visualized in the different cross sections of seminiferous tubules as a semicircular or pie-shaped pattern of staining that was also dependent on the different stages of the cycle.
In the efferent ducts, intense staining for the anti–AQP-10 antibody was noted over the microvilli of the nonciliated cells as well as over the cilia of the ciliated cells (Figure 1e). While there was no reaction over the epithelial cells of the entire epididymis, the endothelium of vascular channels of the intertubular spaces of the efferent ducts and epididymis was intensely reactive (data not shown).
In the testis and efferent ducts, there was no expression of AQP-3 other than background levels of staining. However, in the epididymis, AQP-3 was intensely expressed in the epithelium in a cell-specific manner but was not restricted to any given epididymal region (Figure 2a and b). The cell type that showed an intense immunoperoxidase reaction product was the basal cell (Figure 2a and b). The latter types of cells reside at the base of the epithelium, and the reaction clearly defined the boundaries of these cells, leaving their nuclei completely unstained. The thin lateral processes of basal cells were also stained and at times appeared as thin bands or islands of cytoplasm detached from their main cell body (Figure 2a and b). On occasion, processes of these cells extended toward the lumen but did not contact it (Figure 2b). Throughout the epididymal duct, there was no reaction over the epithelial principal, clear, or narrow cells. Likewise, sperm in the lumen were completely unreactive (Figure 2a).
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During the course of this study, we also examined tissues fixed in Ste Marie fixative as well as frozen sections of weakly fixed paraformaldehyde testicular and epididymal tissue. In addition to the ABC method of immunostaining, we employed microwave antigen retrieval methods, trypsin digestion methods to expose antigenic sites, and routine immunoperoxidase staining methods. In all cases, the staining pattern was identical, but since the ABC method gave the best results, we included only this protocol in the present study. We also employed different dilutions from 1:50 to 1:200, with the 1:100 dilution giving the best results. In addition, when the various tissues were immunostained by omitting the primary antibody or when they were treated with normal rabbit serum, there was no reaction over any area of the tissue, including the epithelium, interstitial space of the testis, efferent ducts, and epididymis, as well as sperm in the lumen (Figure 2a, inset). Furthermore, each antibody gave a staining pattern that differed from one another and from that noted for other AQP antibodies (AQP-1, AQP-8, and AQP-9) published by us, suggesting the specificity of each of these 3 AQP antibodies.
Regulation of AQP-3 in the Epididymis
At the different time intervals after orchidectomy, the epididymal
epithelium was completely devoid of reaction product. In contrast to the
intense staining of basal cells seen in control animals, no reaction was noted
over these cells (Figure 2c).
In 14- and 21-day orchidectomized animals that were supplemented immediately
with high levels of testosterone, a reaction was restored to basal cells;
however, this reaction was not comparable to the degree that was seen in
control animals (Figure 2d).
Efferent duct ligation at the different time intervals also resulted in a loss
of reactivity over basal cells (Figure
2e), but it was not as dramatic as that seen in orchidectomized
animals; however, basal cells were not as reactive as that seen in
testosterone-supplemented animals. Thus, efferent duct ligation showed levels
of reactivity over basal cells that were intermediate to the complete absence
of staining noted in orchidectomized animals and the moderate reaction noted
in testosterone-supplemented orchidectomized animals.
To ensure a consistent and reproducible staining pattern for each antibody, at least 30 slides were examined from each of the 4 control animals. In regulation studies for AQP-3, a similar number was employed for each animal at a given time point of each experimental protocol.
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Discussion |
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In the testis, Sertoli cells carry out a variety of functions, many of which are related to events taking place during spermatogenesis (Russell, 1993). One of their functions is to continuously produce fluid that bathes the developing germ cells, which serves as the vehicle for sperm to enter the epididymis (Setchell et al, 1969; Voglmayr et al, 1970; Hinton and Setchell, 1993). In this regard, expression of AQP-8 already described in Sertoli cells (Badran and Hermo, 2002) would be involved in the transport of water from the interstitial space into the lumen, and this would occur at all stages of the cycle. However, the finding of AQP-0 in these cells and maximally expressed at stages VI–VIII could also assist in this function. Stages VI–VIII correlate with the period of time immediately prior to and precisely when the elongating spermatids are being released into the lumen. Thus, the presence of 2 AQPs at this time point during the cycle may greatly facilitate the transport of water into the lumen and hence the movement of sperm out of the seminiferous tubules. A rationale for the semicircular pattern of AQP-0 expression in Sertoli cells is not at present clear. However, it may be that, at the time of spermiation, not all the elongating spermatids of a given tubule are released simultaneously, thus accounting for the asynchronous staining pattern of AQP-0 in Sertoli cells. Further experimentation involving AQP-0 and its role in Sertoli cells is required.
In the testis, AQP-0 was also noted over numerous polygonal deeply stained interstitial cells. Leydig cells are clustered together and comprise the major cell type of interstitial space—more than 75% (Wing and Christensen, 1982). The other major cell type of this space is the macrophage. In contrast, these cells are pale when stained and are widely scattered in the interstitial space. It is therefore suggested that the cells stained in this space are Leydig cells (Table 1). Leydig cells, steroid-secreting cells, have also been shown to express AQP-9 (Tsukaguchi et al, 1999; Elkjaer et al, 2000; Nihei et al, 2001; Badran and Hermo, 2002). Thus, Leydig cells appear to express more than 1 AQP. While both AQP-9 and AQP-0 may maintain water equilibrium in these cells, AQP-9 is also involved in the passage of purines, pyrimidines, and glycerol (Tsukaguchi et al, 1999).
In the testis, AQP-8, along with AQP-0, now appears to reside in Sertoli cells, and AQP-9, along with AQP-0, appears to reside in Leydig cells. The presence of more than 1 AQP in the same cell type suggests the importance of water transport in that cell or, alternatively, that AQPs serve more than 1 function. The expression of AQP-0, also known as major intrinsic protein-26 (MIP26), has been well documented in the lens fiber of the eye, where it makes up to 80% of the total lens membrane protein (Shiels and Bassnett, 1996). AQP-0 is a 263–amino acid transmembrane protein that contains 6 transmembrane domains, where both the N and C termini are predicted to be cytoplasmic. However, AQP-0 belongs to the family of AQPs that are highly selective for water and not to the aquaglyceroporin family of AQPs (ie, AQP-3, AQP-7, and AQP-9) that, in addition to water, transport glycerol and urea (Verkman and Mitra, 2000). Thus, it remains to be determined why AQP-0 and AQP-8 reside in Sertoli cells.
The efferent ducts have a major role in reabsorbing water that enters the lumen of these ducts from the seminiferous tubules of the testis. In fact, the nonciliated epithelial cells resorb about 50%–90% of the fluid coming from the testis (Crabo, 1965; Setchell and Brooks, 1988; Clulow et al, 1998). In terms of the distribution of members of the AQP family thus far, both AQP-1 and AQP-9 are expressed on the nonciliated cells of the efferent ducts (Brown et al, 1993; Fisher et al, 1998; Pastor-Soler et al, 2001). However, while the anti–AQP-1 antibody decorated the microvilli and basolateral plasma membranes of the epithelial nonciliated cells, the anti–AQP-9 antibody was restricted to the microvilli of the nonciliated cells (Badran and Hermo, 2002). AQP-1 also stained endosomes whereby water could be removed from these structures as they evolved into smaller, denser, and more compact lysosomes (Badran and Hermo, 2002). In the present study, AQP-10 was noted solely on the microvilli of the nonciliated cells, not on their basolateral plasma membranes or endosomes, and it was also detected in the ciliated cells (Table 1), as noted for AQP-1 (Badran and Hermo, 2002). AQP-10, first identified in human absorptive epithelial cells of the small intestine, encodes a 264–amino acid protein with high sequence identity with AQP-3, AQP-7, and AQP-9 (Hatakeyama et al, 2001; Ishibashi et al, 2002). Since AQP-10 appears to be permeable to glycerol (Ishibashi et al, 2002), its presence in the efferent ducts may be related to the transport of glycerol.
In the efferent ducts, water resorption in the nonciliated cells involves an apically located Na+/H+ exchanger, isoform NHE3, that would act as a standing osmotic gradient to move water from the lumen into the cell (Hansen et al, 1999; Bagnis et al, 2001; Leung et al, 2001; Zhou et al, 2001). The apical expression of AQP-1, AQP-9, and AQP-10 in the nonciliated cells would allow the rapid passage of water between these 2 sites. The expression of AQP-1 on the basolateral plasma membrane of these cells would then serve to rapidly remove water from the cell into the intertubular space. The presence of AQP-1, AQP-9, and AQP-10, all decorating the microvilli of the nonciliated cells of the efferent ducts, suggests the need for rapid movement of water across these cells. However, the presence of more than 1 AQP in a given cell type also suggests that these AQPs perform other, as yet unknown functions. It has yet to be demonstrated if the pore sizes of the different AQPs are similar or not; this may influence the passage of large amounts of water across the cell as well as other molecules. The finding of more than 1 AQP in a given tissue is not a rare phenomenon. Indeed, in the kidney and intestine, as examples, several members of the AQP family of proteins have been localized that often show cell-type and region specificity, with more than 1 AQP being present on the same cell type (King and Agre, 1996; Wintour, 1997; Nielsen et al, 2002). Curiously, AQP-10, as well as AQP-1 (Badran and Hermo, 2002), is expressed in the ciliated cells. Thus, these cells, which have also been shown to be endocytic (Hermo et al, 1985), may also aid in the movement of water from the lumen of the efferent ducts to the intertubular space.
As for AQP-1 (Badran and Hermo,
2002), the endothelial cells of vascular channels of the efferent
ducts and epididymis were also stained with the anti–AQP-10 antibody
(Table 1). Thus, once water is
transported across the epithelium, it would move into the vascular channels by
means of both AQP-1 and AQP-10 to maintain water equilibrium in these tissues.
In the efferent ducts, the removal of water concentrates sperm in the lumen of
the initial segment, which is small compared to the lumen of the remaining
epididymal regions (Hamilton, 1975). This would facilitate the interactions of
the secretory products of principal cells with the sperm surface and thus
enable them to acquire their maturational characteristics
(Cooper, 1995). The
infertility of male mice in the ERKO mouse model system, which results
in the retention of water and a diluted sperm concentration in the initial
segment, is indicative of the importance of water removal in the efferent
ducts (Lubahn et al, 1993; Eddy et al, 1996;
Hess et al, 1997;
Zhou et al, 2001).
Although AQP-10 was present in the efferent ducts, it was not expressed in the testis or epididymal epithelium. In the epididymal epithelium, AQP-9 has been localized to the microvilli of principal cells, with the most intense reaction being noted in the initial segment and cauda regions (Elkjaer et al, 2000; Pastor-Soler et al, 2001; Badran and Hermo, 2002). In addition, AQP-9 was expressed in clear cells but only in those of the cauda region (Badran and Hermo, 2002). Thus, the expression of AQP-9 appears to be region-specific in principal and clear cells, with no expression of AQP-1 or AQP-8 in the epididymal epithelium (Badran and Hermo, 2002).
In the present study, AQP-3 was detected only in the epididymis and not in the testis or efferent ducts. The cell type that was reactive was the basal cell, and this was noted in all epididymal regions (Table 1). In the epididymis, water continues to be reabsorbed from the lumen (Levine and Marsh, 1971; Setchell and Brooks, 1988; Wong et al, 2002). The finding of AQP-3 in basal cells suggests that there is an intricate cooperative removal of water from the epididymal lumen that appears to involve the different cell types. For example, in the initial segment, AQP-9 is found on principal cells, and AQP-1 is found on myoid cells enveloping the tubules and endothelial cells of vascular channels (Badran and Hermo, 2002). The present finding of AQP-3 on basal cells suggests that water is rapidly transported from the epididymal lumen across the entire width of the epithelium, including the myoid cell layers, to eventually reach the lumen of vascular channels. AQP-3 expression in basal cells does not appear to be unique to the epididymis. Indeed, basal cells of the epithelium of the trachea also express AQP-3 (Nielsen et al, 1997). In addition, AQP-3 has been demonstrated in various cell types and regions of the kidney and gastrointestinal tract (Ishibashi et al, 1997b; Schrier and Cadnapaphornchai, 2003).
AQP-3 is a 31.4-kd protein, with 285 amino acids, that plays a major role in water and urea exit mechanisms in the collecting duct cells of the kidney. It is also highly permeable to glycerol and is hence a member of the aquaglyceroporin family of proteins (Ishibashi et al, 1994, 1997a). Thus, while AQP-3 in basal cells of the epididymis may move water across the epithelium, it may also transport glycerol. Indeed, glycerol is a component of the epididymal fluid at concentrations of about 1.15 mM, with epididymal sperm using glycerol to produce CO2 (Cooper and Brooks, 1981). Glycerol is derived from glycerylphosphorylcholine (GPC) through the activity of GPC cholinephosphohydrolase. GPC is found at high concentrations in epididymal fluids and the epididymis (Dawson and Rowlands, 1958), where it is presumably synthesized within principal and basal cells and transported to the lumen, where, in turn, it will be used by sperm (Killian and Chapman, 1980). Thus, the presence of AQP-3 in basal cells, along with the expression of AQP-9 in principal cells, may lead to the efficient transport of glycerol, as well as GPC, from the epithelium to the epididymal lumen, where they could play a functional role in relation to sperm maturation.
Basal cells reside in a variety of different epithelial tissues, but these cells have not been studied in much detail. Recent findings on these cells in the epididymis suggest that they have a unique structural appearance and perform a variety of functions (Veri et al, 1993). In the adult epididymis, these cells do not divide and thus are not stem cells (Robaire and Hermo, 1988). In this tissue, basal cells are small, hemispherical cells residing on the basement membrane and are not in apparent contact with the lumen of the duct, even though, on occasion, they send thin processes apically. In addition, basal cells possess attenuated, thin, footlike processes that extend along the basement membrane in such a way as to collectively encompass a large portion of the circumference of each tubule (Veri et al, 1993). Thus, they form a barrier, albeit an incomplete one, between the epididymal lumen, on the one hand, and the blood vessels and other contents of the intertubular space, on the other. Therefore, to a degree, they can effectively eliminate potentially harmful substances emanating from the blood, trying to access the sperm in the lumen. In this context, basal cells express various antioxidants (Nonogaki et al, 1992; Papp et al, 1995) and metallothioneins (Cyr et al, 2001). Basal cells also express connexin-43 with neighboring principal cells, a gap junctional protein by which these 2 cell types can communicate information with one another (Cyr et al, 1996). The finding of AQP-3 expression adds another twist to the ever-growing functions that basal cells perform that now include the transport of water and glycerol transport in the epididymal epithelium.
Regulation of AQP-3 in the Epididymis
In the present study, an examination of the regulation of AQP-3 expression
in the epididymis revealed that basal cells became unreactive in the absence
of testicular factors (Table
2). The intensity of the reaction with the anti–AQP-3
antibody was also considerably reduced after efferent duct ligation,
suggesting that circulating levels of testosterone alone could not maintain
AQP-3 expression at control levels and that a luminal factor emanating from
the testis was also needed. This was confirmed in orchidectomized animals that
were supplemented with high levels of testosterone. In such cases, the
reaction was enhanced above that seen for efferent duct ligated animals, but
not to the degree that is seen in control animals. It was thus suggested that
AQP-3 expression in basal cells was dependent, in part, on testosterone and,
in part, on a luminal testicular factor. In comparison, regulation studies of
AQP-9 in the adult rat epididymis revealed cell-type and region specificity.
In the initial segment, where it was intensely expressed on the microvilli of
principal cells in controls, a dependence of both testosterone and a lumicrine
factor was noted for maximal expression
(Badran and Hermo, 2002). A
similar situation was also reported for clear cells of the cauda epididymidis
(Badran and Hermo, 2002). It
remains to be determined whether or not the lumicrine factor regulating AQP-9
and AQP-3 expression in principal and clear cells and in basal cells,
respectively, is the same or different for each cell type. Nevertheless, AQP-9
staining in principal cells of the caput, corpus, and cauda regions was not
modified from controls after efferent duct ligation or orchidectomy,
suggesting no dependence on testicular factors for its expression in these
regions (Badran and Hermo,
2002). Thus, while there are some similarities in the pattern of
regulation for AQP-9 and AQP-3 in the various epithelial cell types,
differences are also apparent.
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In the epididymis, various factors regulate epididymal functions at both the gene and protein levels (Robaire and Hermo, 1988; Orgebin-Crist, 1996; Cornwall et al, 2002; Ezer and Robaire, 2002). This includes the dependence of androgens on some epididymal functions but not on others (Cornwall et al, 2002; Ezer and Robaire, 2002). However, in addition to the regulation mediated by androgens, factors emanating from the testis that enter the epididymis via the lumen of the duct, defined as lumicrine factors, play a role in regulating epididymal functions (Hinton et al, 1998; Cornwall et al, 2002). Indeed, ligation of the efferent ducts induces changes in epididymal structure and gene and protein expression (Cornwall et al, 2002). Lumicrine factors derived from the testis regulate several proteins synthesized by the epididymis (Garrett et al, 1991; Lareyre et al, 2001; Cornwall et al, 2002; Hermo and Andonian, 2003).
Information regarding the regulation of the function of basal cells is
scanty. In their expression of Yb1-glutathione
S-transferase (GST), basal cells show a differential region-specific
response. In the corpus region, Yb1-GST expression in basal cells
is regulated by testosterone, but in the proximal initial segment, expression
is regulated by a lumicrine factor
(Andonian and Hermo, 2003). These data differ dramatically from that obtained for the Yf-GST subunit,
where its expression in basal cells was unaltered after orchidectomy, efferent
duct ligation, or hypophysectomy, indicating that neither testicular nor
pituitary factors governed Yf-GST expression in basal cells
(Hermo and Papp, 1996). Thus,
basal cells of different regions show differential responses to the absence of
androgens or testicular lumicrine factors in their expression of a given GST
as well as between different GSTs. The expression of metallothionein by basal
cells, although detected in all epididymal regions, was shown to be
androgen-dependent according to specific regions
(Cyr et al, 2001). In the
present study, basal cell expression of AQP-3 appears to be dependent on both
testosterone and a lumicrine factor. Although this is the first demonstration
of the dependence of regulating factors of different origins on basal cells,
at the messenger RNA level, 
-glutamyl transpeptidases, which show
multiple transcripts, have been shown to be differentially regulated by
androgens and/or lumicrine factors in the different epididymal regions
(Palladino and Hinton, 1994).
Thus, the dependence of several regulating factors is not uncommon for
epididymal cell functions.
In summary, the present study indicates that expression of AQP-0, AQP-3, and AQP-10 is cell-, tissue-, and region-specific in the testis, efferent ducts, and epididymis of adult rats. In addition, both testosterone and a lumicrine factor appear to regulate the expression of AQP-3 in basal cells of the epididymis.
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Footnotes |
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References |
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