Region- and Cell-specific Differences in the Distribution of Carbonic Anhydrases II, III, XII, and XIV in the Adult Rat Epididymis
Department of Anatomy and Cell Biology (LH) and Faculty of Dentistry (DLC), McGill University, Montreal, Quebec; Departement de Stomatologie, Universite de Montreal, Montreal, Quebec (PM,CES); and Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri (WSS,AW)
Correspondence to: Dr. Louis Hermo, McGill University Department of Anatomy and Cell Biology, 3640 University St., Montreal, Quebec, Canada H3A 2B2. E-mail: louis.hermo{at}mcgill.ca
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Summary |
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Key Words: RT-PCR immunocytochemistry principal cells carbonic anhydrase epididymis pH basal cells region specificity rat
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Introduction |
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In the lumen of the epididymal duct, the concentrations of ions such as Na+ decline from the caput to the cauda epididymidis, whereas HCO3 ions show a sharp decline through the initial segment followed by a slight rise as pH changes from a starting value near pH 7.2 to a low of pH 6.6 followed by a slight rise to pH 6.8 in the cauda (Levine and Marsh 1971; Levine and Kelly 1978
; Turner 1984
; Caflisch and DuBose 1990
; Turner 2002
; Breton 2003
). It has been shown in several species that this mild fluid acidification immobilizes cauda sperm, and this prevents premature activation of their acrosomal enzymes in the epididymis (Acott and Carr 1984
; Carr et al. 1985
; Clulow et al. 1992
; Gatti et al. 1993
; Hinton et al. 1995
; Jones and Murdoch 1996
). Although exact details of the mechanisms of this acidification are not fully understood, the enzyme CA is clearly involved (Cohen et al. 1976
; Brown et al. 1992
Kaunisto et al. 1995
; Breton et al. 1999
; Hermo et al. 2000
; Breton 2003
), as are apically positioned epithelial H+-ATPase proton pumps (Breton et al. 1996
; Hermo et al. 2000
).
CAs exist as three genetically unrelated families of proteins, of which only the genes are present in vertebrates (Chegwidden and Carter 2000
; Sly 2000
). The
-CAs are monomeric zinc metalloenzymes of
29-kDa molecular mass. So far, several different
-CA isoforms have been reported (Vince and Reithmeier 1998
; Chegwidden and Carter 2000
; Parkkila 2000
; Sly 2000
; Tripp et al. 2001
). CAs are important for acidbase regulation, catalyzing the reversible reaction of CO2 + H2O to H+ + HCO3. Several CAs can be expressed in the same mammalian tissue, and although they may show cell type, region, and subcellular specificity, more than one CA can be expressed in the same cell type. CAs have unique functions in different cellular locations and result in a variety of diseases in their alteration or absence (Chegwidden and Carter 2000
; Parkkila 2000
; Sly 2000
; Tripp et al. 2001
). Whereas some CAs are present in the cytoplasm (IIII, VII, XIII), others are found in mitochondria (V) or salivary secretions (VI). The remaining CAs (CA IV, IX, XII, and XIV) are transmembrane proteins, with CA IV being glycosylphosphatidylinositol (GPI) anchored (Chegwidden and Carter 2000
; Parkkila 2000
; Sly 2000
; Tripp et al. 2001
).
In the adult rat efferent ducts and epididymis, only two isoforms of the CA family of proteins have been investigated to date. In the epididymis, CA IV has been well documented to be present along the apical plasma membranes of principal cells in the distal caput, the corpus, and proximal cauda regions, with maximal expression occurring in the corpus (Kaunisto et al. 1995,1999
). However, results for CA II distribution have been conflicting, with localizations reported in narrow cells of the initial segment and intermediate zone (Hermo et al. 2000
), both narrow and clear cells (Breton et al. 1996
,1998
) and narrow and principal cells (Kaunisto et al. 1995
,1999
). CA II has also been identified in the epithelial non-ciliated cells of efferent ducts (Hess 2002
).
The epithelial cells of the different segments of the kidney nephron bear notable structural and functional similarities to those of the efferent ducts and epididymis on the basis of embryological derivations, with the proximal convoluted tubule bearing similarity to the efferent ducts and proximal epididymal regions and the collecting duct to the distal epididymal regions and the vas deferens (Hinton and Turner 1988; Hess 2002
). In the kidney, many CA isoforms have been identified and are often expressed in a precise cell- and region-specific manner including CA II, IV, XII, and XIV (Mori et al. 1999
; Sly 2000
; Sterling et al. 2001
; Kaunisto et al. 2002
; Kyllonen et al. 2003
). On this basis, it seemed reasonable to hypothesize that isoforms other than CA II and IV are probably expressed in the efferent ducts and epididymis of adult rats. This was investigated by RT-PCR using oligonucleotide primers specific for rat CA II and IV as positive controls and other isoform-specific primers for CAs known to be present in kidney and other cell types. RT-PCR results for identified isoforms were complemented with light microscope immunocytochemistry.
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Materials and Methods |
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Dako Envision+ System Kit
Primary antibodies used in this study were mouse monoclonal CA III (1:100) (courtesy of Spectral Diagnostics; Toronto, ON, Canada), rabbit anti-mouse CA XII (1:100) (Tureci et al. 1998), rabbit anti-mouse CA XIV (1:100) (Mori et al. 1999
), and rabbit anti-bovine CA II antibody (Biodesign; Saco, ME).
Sections of Bouin's and St. Marie's fixed tissues were deparaffinized with Histoclear and rehydrated in a series of 100%, 100%, 95%, 80%, 70%, and 50% ethanol solutions and distilled water, respectively. During hydration, residual picric acid was neutralized by using a 70% ethanol solution containing 1% lithium carbonate. After hydration, endogenous peroxidase activity was blocked for 5 min with Dako peroxidase blocking reagent (DakoCytomation; Mississauga, ON, Canada) followed by washing. Localizations of CA II, III, XII, and XIV were performed using the Dako Envision+ Peroxidase diaminobenzidine (DAB) kit. Washings between each step were done for 10 min using a buffer solution containing 0.05 M Tris, 0.3 M NaCl, and 0.1% Tween 20, pH 7.27.6. Substratechromogen solutions were prepared by adding one drop of liquid DAB + chromogen with an additional 4 µl to 1 ml of the buffered substrate. The sections were counterstained for 10 sec in a 1:5 diluted solution of 0.1% methylene blue and 0.1% thionin, washed, and quickly back dehydrated to Histoclear. Coverslips were then mounted with Permount.
Immunofluorescence
Primary antibodies used were rabbit anti-mouse CA XII (1:100) (Tureci et al. 1998) and rabbit anti-mouse CA XIV (1:100) (Mori et al. 1999
); the secondary antibody was an AlexaFluor 594-labeled goat anti-rabbit IgG (1:250) (Molecular Probes; Eugene, OR).
Sections of St. Marie's fixed tissues were deparaffinized as per the DAKO protocol. Blocking of nonspecific binding sites was done on some sections using the Dako protein block serum-free solution for 20 min (DakoCytomation), followed by incubations with primary antibody for 1.5 hr at room temperature. The sections were washed and incubated for 30 min with AlexaFluor 594-labeled goat anti-rabbit IgG. Following this step, nuclei were stained by incubating the sections for 5 min in a 300-nM 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) solution at room temperature. Coverslips were mounted using Vectashield Hard Set aqueous mounting medium (Vector Laboratories; Burlington, ON, Canada). The sections were examined and photographed on a Zeiss Axioskop 2 motorized light microscope equipped with variable intensity FluorArc epifluoresence mercury lighting and AxioCam HR color digital camera (Carl Zeiss Canada; Montreal, QC, Canada).
Negative controls were performed for each experiment and consisted of exposing sections to solutions containing all components except primary antibody (polyclonal and monoclonal) or normal rabbit serum (polyclonal antibodies). Positive controls were also done using sections from tissues known to express specific CA isoforms (e.g., CA III, skeletal muscle; CA IV, kidney; CA XIV, liver).
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Results |
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Sections incubated with the anti-CA II antibody also showed reactions over the non-ciliated and ciliated cells of the efferent ducts (not shown). Narrow cells of the initial segment were intensely reactive, as were principal cells across the entire epididymis, with the reaction being cytoplasmic (Figures 4A and 4B). Clear and basal cells of the entire epididymis were consistently unreactive (Figures 4A and 4B). However, the cytoplasmic droplets of sperm in the epididymal lumens showed an intense reaction (Figure 4B), as did the cytoplasm of elongating spermatids of the seminiferous epithelium in the testis (not shown). Smooth muscle cells of the cauda epididymidis were also intensely reactive with the anti-CA II antibody (not shown).
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Discussion |
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The distribution of CA II in the rat epididymis is a matter of dispute among investigators. Aside from universal agreement regarding its localization to narrow cells of the initial segment, it has been suggested that CA II is expressed in clear cells and principal cells (Kaunisto et al. 1995,1999
; Breton et al. 1996
; Hermo et al. 2000
). In the present study, CA II was localized to narrow cells of the initial segment and to principal cells of all regions, except the distal cauda, where its expression was patchy, but on no occasion was CA II expression found in clear cells (Figure 8).
In the present study, CA III was localized to principal cells of the epididymis but not to narrow or clear cells (Figure 8). CA III, like CA II, is cytosolic, suggestive of redundancy of functions in principal cells. However, CA II is an enzyme of much higher efficiency than CA III, performing functions in respiration and acidbase balance, and is more widely expressed in mammalian cells than is CA III. Interestingly, the CA II knockout mouse model does not have a phenotype (males are fertile; Zhou et al. 2001), and this may be attributed to the fact that CA III is distributed throughout the epididymis in the same cells that express CA II.
In addition to carrying out reversible hydration reactions, CA III has two free thiols that scavenge oxygen radicals during oxidative stress (Cabiscol and Levine 1995). In the epididymis, the luminal environment is oxygen rich, and unsaturated fatty acids present in sperm membranes are susceptible to oxidative damage (Hinton et al. 1995
). Sperm generate reactive oxygen species, such as superoxide anion (O2) and hydrogen peroxide (H2O2), especially in the cauda region, where the sperm are stored (Alvarez and Storey 1982
,1984
; Aitken 2002
). To this end, principal cells throughout the epididymis protect sperm from harmful free radical injury by expressing numerous isoforms of glutathione S-transferase (GST), gamma glutamyl transferases, and other free radical scavengers (Palladino and Hinton 1994
; Papp et al. 1995
; Aitken 2002
). Thus, expression of CA III in the epididymis, as well as in the efferent ducts, may also serve in a protective role for sperm. Interestingly, although basal cells express various GSTs, these cells did not express CA III. On the other hand, clear cells do not express GSTs (Papp et al. 1995
), and these cells also showed no staining for CA III or any other CA isoform in the present study (Figure 8).
CA XII appears to be maximally expressed in the corpus and proximal cauda epididymidis, where it is localized to the basolateral plasma membranes of adjacent principal cells (Figure 8), corresponding to similar localizations in analogous regions of the kidney (Parkkila 2000). Weak staining for CA XII in proximal convoluted tubules (Parkkila 2000
) correlates with similar expression in the efferent ducts, inasmuch as both of these ducts derive from similar embryological origins. CA XII has been localized in varying degrees to the efferent ducts and epididymis of humans (Karhumaa et al. 2001
).
In the epididymis, the vacuolar H+-ATPase pump has been localized apically to narrow cells of the initial segment (Breton et al. 1996,1998
; Hermo et al. 2000
). Together with CA II expression in these cells, it has been suggested that these two players could be involved in acidification of the epididymal lumen of this region to help maintain sperm in a quiescent state as they acquire the capacity for motility during their transit down the duct (Carr et al. 1985
; Hinton et al. 1995
; Jones and Murdoch 1996
). In addition, narrow cells express CA XII basolaterally (Figure 8), which together with basolateral expression of AE2, NBC1, and NBC3 (Jensen et al. 1999a
,b
; Pushkin et al. 2000
), presumably assists in fine-tuning the pH of these cells and their extracellular environment. Interestingly, clear cells express vacuolar H+-ATPase (Breton et al. 1996
, 1998
; Hermo et al. 2000
), yet no CA isoform has been identified within these cells (Figure 8).
Basal cells of the corpus and proximal cauda regions show maximal expression of CA XII (Figure 8), and AQP-3 is also known to be present in the membranes of these cells (Hermo et al. 2004). Halo cells of the initial segment expressed CA XII, but no data exist on associated ion transporters. Considered as monocytes and/or lymphocytes (Robaire and Hermo 1988
; Flickinger et al. 1997
; Serre and Robaire 1999
), halo cells, like basal cells, presumably fine-tune the pH of their respective environments by means of CA XII.
CA XIV, a transmembrane protein, highlighted apical and/or basal plasma membrane domains of principal cells and adjacent areas of their cytosol in a region-specific manner, with sporadic region-specific lateral plasma membrane reactions (Figure 8). The apical and basal cytosolic reactions may indicate the presence of CA XIV within vesicles destined for apical or basal plasma membrane domains, although electron microscopy was not performed. CA IV, a GPI-anchored protein, has already been localized to the apical plasma membranes of principal cells of the distal caput, corpus, and proximal cauda regions of the rat epididymis, with maximal staining in the corpus region, and this correlates with similar distribution in the human epididymis (Parkkila et al. 1993; Kaunisto et al. 1995
,1999
). The present finding of CA XIV mainly in apical areas of principal cells of proximal epididymal regions suggests that CA XIV functions in these regions, rather than CA IV, which resides more distally. In distal regions, CA XIV resides both apically and basally in association with principal and basal cells (Figure 8). The intensity of CA XIV expression along the base of the epithelium at distal epididymal sites correlates with the presence of CA XII in distal regions, in both principal cells and basal cells, suggesting redundancy of CA functions or the fact that each may perform slightly different functions.
The staining intensity for CA III in principal cells of the distal cauda epididymidis is variable and not apparent in every cell. Similarly, basolateral CA XII expression in principal cells of the distal initial segment and caput epididymidis is patchy, as is that for basolateral expression of CA XIV in principal cells of proximal regions. This type of staining pattern is not uncommon for the epididymis. Indeed, expression of many proteins, including secretory, lysosomal, and protective proteins, occurs in a patchy manner, described as a checkerboard staining pattern whereby a given cell type in a given cross-sectional profile of tubule shows varying degrees of reactivity or complete unreactivity for a given protein (Hermo et al. 1991,1992
,1994
,1998
; Rankin et al. 1992
; Veri et al. 1993
; Papp et al. 1995
). It has been suggested that cells showing this pattern of reactivity may not be expressing a particular protein of interest, or that they may be out of synchrony with one another in expressing it (Hermo et al. 1991
). The significance of such a phenomenon has yet to be resolved, but it clearly extends to the CAs themselves.
Establishing an acidic pH and a low bicarbonate concentration in the epididymal lumen keeps sperm in a quiescent state (Babcock et al. 1983; Acott and Carr 1984
; Okamura et al. 1985
; Tajima et al. 1987
; Holm and Wishart 1998
; Gatti et al. 1993
). Changes in bicarbonate composition in the lumen trigger sperm motility and activation of their acrosomal enzymes, leading to sperm death (Lee and Storey 1986
). Altering the ionic concentration of the luminal fluid has significant effects on epithelial secretion of proteins and other compounds, which could affect sperm maturation (Au and Wong 1980
). Thus, monitoring the proper ionic composition of the epididymal lumen in a sequential manner down the duct has relevance to sperm maturation and viability. The coordinated activities of the various epithelial cells of the epididymis, by expressing or coexpressing different CA family members of proteins in conjunction with various channels and transporters in a cell type, subcellular, and region-specific manner, must ultimately lead to the desired pH of the epididymal lumen essential for proper sperm function.
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Acknowledgments |
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We thank Victoria Smith and Neelum Jamal for technical assistance during the course of this study. We also thank Haitham Badran for preparing the schematic drawing shown in Figure 8.
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Footnotes |
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Acott TS, Carr DW (1984) Inhibition of bovine spermatozoa by caudal epididymal fluid: II. Interaction of pH and a quiescence factor. Biol Reprod 30:926935
Aitken RJ (2002) Active oxygen in spermatozoa during epididymal transit. In Robaire B, Hinton BT, eds. The Epididymis: From Molecules to Clinical Practice. New York, Kluwer Academic/Plenum Publishers, 435448
Alvarez JG, Storey BT (1982) Spontaneous lipid peroxidation in rabbit epididymal spermatozoa: its effect on sperm motility. Biol Reprod 27:11021108
Alvarez JG, Storey BT (1984) Lipid peroxidation and the reactions of superoxide and hydrogen peroxide in mouse spermatozoa. Biol Reprod 30:833841
Au CL, Wong PY (1980) Luminal acidification by the perfused rat cauda epididymidis. J Physiol 309:419427[Medline]
Babcock DF, Rufo GA Jr, Lardy HA (1983) Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm. Proc Natl Acad Sci USA 80:13271331
Badran HH, Hermo LS (2002) Expression and regulation of aquaporins 1, 8, and 9 in the testis, efferent ducts, and epididymis of adult rats and during postnatal development. J Androl 23:358373
Bagnis C, Marsolais M, Biemesderfer D, Laprade R, Breton S (2001) Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis. Am J Physiol Renal Physiol 280:F426F436
Breton S (2003) Luminal acidification in the epididymis and vas deferens. In Hinton BT, Turner TT, eds. Third International Conference on the Epididymis. Charlottesville, VA, The Van Doren Company, 6072
Breton S, Hammar K, Smith PJ, Brown D (1998) Proton secretion in the male reproductive tract: involvement of Cl-independent HCO-3 transport. Am J Physiol 275:C1134C1142[Medline]
Breton S, Smith PJ, Lui B, Brown D (1996) Acidification of the male reproductive tract by a proton pumping (H+)-ATPase. Nat Med 2:470472[CrossRef][Medline]
Breton S, Tyszkowski R, Sabolic I, Brown D (1999) Postnatal development of H+ ATPase (proton-pump)-rich cells in rat epididymis. Histochem Cell Biol 111:97105[CrossRef][Medline]
Brown D, Lui B, Gluck S, Sabolic I (1992) A plasma membrane proton ATPase in specialized cells of rat epididymis. Am J Physiol 263:C913C916[Medline]
Cabiscol E, Levine RL (1995) Carbonic anhydrase III. Oxidative modification in vivo and loss of phosphatase activity during aging. J Biol Chem 270:1474214747
Caflisch CR, DuBose TD Jr (1990) Direct evaluation of acidification by rat testis and epididymis: role of carbonic anhydrase. Am J Physiol 258:E143E150[Medline]
Carr DW, Usselman MC, Acott TS (1985) Effects of pH, lactate, and viscoelastic drag on sperm motility: a species comparison. Biol Reprod 33:588595
Chegwidden WR, Carter ND (2000) Introduction to the carbonic anhydrases. In Chegwidden WR, Carter ND, Edwards YH, eds. The Carbonic Anhydrases. Basel, Switzerland, Birkhauser Verlag, 1328
Clulow J, Jones RC, Hansen LA, Man SY (1998) Fluid and electrolyte reabsorption in the ductuli efferentes testis. J Reprod Fertil 53(suppl):114
Clulow J, Jones RC, Murdoch RN (1992) Maturation and regulation of the motility of spermatozoa in the epididymis of the tammar wallaby (Macropus eugenii). J Reprod Fertil 94:295303[Medline]
Cohen JP, Hoffer AP, Rosen S (1976) Carbonic anhydrase localization in the epididymis and testis of the rat: histochemical and biochemical analysis. Biol Reprod 14:339346[Medline]
Crabo B (1965) Studies on the composition of epididymal content in bulls and boars. Acta Vet Scand 6:894
Cyr DG, Hermo L, Egenberger N, Mertineit C, Trasler JM, Laird DW (1999) Cellular immunolocalization of occludin during embryonic and postnatal development of the mouse testis and epididymis. Endocrinology 140:38153825
Elkjaer M, Vajda Z, Nejsum LN, Kwon T, Jensen UB, Miry-Moghaddam M, Frokiaer J, et al. (2000) Immunolocalization of AQP9 in liver, epididymis, testis, spleen, and brain. Biochem Biophys Res Commun 276:11181128[CrossRef][Medline]
Fisher JS, Turner KJ, Fraser HM, Saunders PT, Brown D, Sharpe RM (1998) Immunoexpression of aquaporin-1 in the efferent ducts of the rat and marmoset monkey during development, its modulation by estrogens, and its possible role in fluid resorption. Endocrinology 139:39353945
Flickinger CJ, Bush LA, Howards SS, Herr JC (1997) Distribution of leukocytes in the epithelium and interstitium of four regions of the Lewis rat epididymis. Anat Rec 248:380390[CrossRef][Medline]
Gatti JL, Chevrier C, Paquignon M, Dacheux JL (1993) External ionic conditions, internal pH and motility of ram and boar spermatozoa. J Reprod Fertil 98:439449[Medline]
Hamilton DW (1975) Structure and function of the epithelium lining the ductuli efferentes, ductus epididymis and ductus deferens in the rat. In Hamilton DW, Greep RO, eds. Handbook of Physiology, vol. 5, sec. 7. Baltimore, Williams and Wilkins, 259301
Hermo L, Adamali HI, Andonian S (2000) Immunolocalization of CA II and H+ V-ATPase in epithelial cells of the mouse and rat epididymis. J Androl 21:376391
Hermo L, Barin K, Oko R (1998) Androgen binding protein secretion and endocytosis by principal cells in the adult rat epididymis and during postnatal development. J Androl 19:527541
Hermo L, Krzeczunowicz D, Ruz R (2004) Cell specificity of aquaporins 0, 3, and 10 expressed in the testis, efferent ducts, and epididymis of adult rats. J Androl 25:494505
Hermo L, Oko R, Morales CR (1994) Secretion and endocytosis in the male reproductive tract: a role in sperm maturation. Int Rev Cytol 154:106189[Medline]
Hermo L, Oko R, Robaire B (1992) Epithelial cells of the epididymis show regional variations with respect to the secretion of endocytosis of immobilin as revealed by light and electron microscope immunocytochemistry. Anat Rec 232:202220[CrossRef][Medline]
Hermo L, Robaire B (2002) Epididymal cell types and their functions. In Robaire B, Hinton BT, eds. The Epididymis: From Molecules to Clinical Practice. New York, Kluwer Academic/Plenum Publishers, 81102
Hermo L, Wright J, Oko R, Morales CR (1991) Role of epithelial cells of the male excurrent duct system of the rat in the endocytosis or secretion of sulfated glycoprotein-2 (clusterin). Biol Reprod 44:11131131
Hess RA (2002) The efferent ductules: structure and functions. In Robaire B, Hinton BT, eds. The Epididymis: From Molecules to Clinical Practice. New York, Kluwer Academic/Plenum Publishers, 4980
Hess RA, Bunick D, Lee KH, Bahr J, Taylor JA, Korach KS, Lubahn DB (1997) A role for oestrogens in the male reproductive system. Nature 390:509512[CrossRef][Medline]
Hess RA, Zhou Q, Nie R (2002) The role of estrogens in the endocrine and paracrine regulation of the efferent ductules, epididymis and vas deferens. In Robaire B, Hinton BT, eds. The Epididymis: From Molecules to Clinical Practice. New York, Kluwer Academic/Plenum Publishers, 317338
Hinton B, Setchell B (1993) Fluid secretion and movement. In Russell LD, Griswold MD, eds. The Sertoli Cell. Clearwater, FL, Cache River Press, 249267
Hinton BT, Palladino MA, Rudolph D, Labus JC (1995) The epididymis as protector of maturing spermatozoa. Reprod Fertil Dev 7:731745[Medline]
Hinton BT, Turner TT (1988) Is the epididymis a kidney analog? News Physiol Sci 3:2831
Holm L, Wishart GJ (1998) The effect of pH on the motility of spermatozoa from chicken, turkey and quail. Anim Reprod Sci 54:4554[CrossRef][Medline]
Jensen LJ, Schmitt BM, Berger UV, Nsumu NN, Boron WF, Hediger MA, Brown D, et al. (1999a) Localization of sodium bicarbonate cotransporter (NBC) protein and messenger ribonucleic acid in rat epididymis. Biol Reprod 60:573579
Jensen LJ, Stuart-Tilley AK, Peters LL, Lux SE, Alper SL, Breton S (1999b) Immunolocalization of AE2 anion exchanger in rat and mouse epididymis. Biol Reprod 61:973980
Jones RC, Murdoch RN (1996) Regulation of the motility and metabolism of spermatozoa for storage in the epididymis of eutherian and marsupial mammals. Reprod Fertil Dev 8:553568[Medline]
Karhumaa P, Kaunisto K, Parkkila S, Waheed A, Pastorekova S, Pastorek J, Sly WS, et al. (2001) Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts. Mol Hum Reprod 7:611616
Kaunisto K, Fleming RE, Kneer J, Sly WS, Rajaniemi H (1999) Regional expression and androgen regulation of carbonic anhydrase IV and II in the adult rat epididymis. Biol Reprod 61:15211526
Kaunisto K, Parkkila S, Parkkila AK, Waheed A, Sly WS, Rajaniemi H (1995) Expression of carbonic anhydrase isoenzymes IV and II in rat epididymal duct. Biol Reprod 52:13501357[Abstract]
Kaunisto K, Parkkila S, Rajaniemi H, Waheed A, Grubb J, Sly WS (2002) Carbonic anhydrase XIV: luminal expression suggests key role in renal acidification. Kidney Int 61:21112118[CrossRef][Medline]
Kaunisto KM, Rajaniemi HJ (2002) Expression and localization of the Na+/H+ exchanger isoform NHE3 in the rat efferent ducts. J Androl 23:237241
Kyllonen MS, Parkkila S, Rajaniemi H, Waheed A, Grubb JH, Shah GN, Sly WS, et al. (2003) Localization of carbonic anhydrase XII to the basolateral membrane of H+-secreting cells of mouse and rat kidney. J Histochem Cytochem 51:12171224
Lee MA, Storey BT (1986) Bicarbonate is essential for fertilization of mouse eggs: mouse sperm require it to undergo the acrosome reaction. Biol Reprod 34:349356
Levine N, Kelly H (1978) Measurement of pH in the rat epididymis in vivo. J Reprod Fertil 52:333335[Medline]
Levine N, Marsh DJ (1971) Micropuncture studies of the electrochemical aspects of fluid and electrolyte transport in individual seminiferous tubules, the epididymis and the vas deferens in rats. J Physiol 213:557570[Medline]
Mori K, Ogawa Y, Ebihara K, Tamura N, Tashiro K, Kuwahara T, Mukoyama M, et al. (1999) Isolation and characterization of CA XIV, a novel membrane-bound carbonic anhydrase from mouse kidney. J Biol Chem 274:1570115705
Okamura N, Tajima Y, Soejima A, Masuda H, Sugita Y (1985) Sodium bicarbonate in seminal plasma stimulates the motility of mammalian spermatozoa through direct activation of adenylate cyclase. J Biol Chem 260:96999705
Palladino MA, Hinton BT (1994) Expression of multiple gamma-glutamyl transpeptidase messenger ribonucleic acid transcripts in the adult rat epididymis is differentially regulated by androgens and testicular factors in a region-specific manner. Endocrinology 135:11461156[Abstract]
Papp S, Robaire B, Hermo L (1995) Immunocytochemical localization of the Ya, Yc, Yb1, and Yb2 subunits of glutathione S-transferases in the testis and epididymis of adult rats. Microsc Res Tech 30:123[CrossRef][Medline]
Parkkila S (2000) An overview of the distribution and function of carbonic anhydrase isozymes in mammals. In Chegwidden WR, Carter ND, Edwards YH, eds. The Carbonic Anyhydrases. Basel, Switzerland, Birkhauser Verlag, 7994
Parkkila S, Parkkila AK, Kaunisto K, Waheed A, Sly WS, Rajaniemi H (1993) Location of a membrane-bound carbonic anhydrase isoenzyme (CA IV) in the human male reproductive tract. J Histochem Cytochem 41:751757
Pastor-Soler N, Bagnis C, Sabolic I, Tyszkowski R, McKee M, Van Hoek A, Breton S, et al. (2001) Aquaporin 9 expression along the male reproductive tract. Biol Reprod 65:384393
Pushkin A, Clark I, Kwon TH, Nielsen S, Kurtz I (2000) Immunolocalization of NBC3 and NHE3 in the rat epididymis: colocalization of NBC3 and the vacuolar H+-ATPase. J Androl 21:708720
Rankin TL, Ong DE, Orgebin-Crist M-C (1992) The 18-kDa mouse epididymal protein (MEP 10) binds retinoic acid. Biol Reprod 46:767771
Robaire B, Hermo L (1988) Efferent ducts, epididymis, and vas deferens: structure, functions, and their regulation. In Knobil E, Neill JD, eds. The Physiology of Reproduction. New York, Raven Press, 9991080
Serre V, Robaire B (1999) Distribution of immune cells in the epididymis of the aging Brown Norway rat is segment-specific and related to the luminal content. Biol Reprod 61:705714
Sly WS (2000) The membrane carbonic anhydrases: from CO2 transport to tumor markers. In Chegwidden WR, Carter ND, Edwards YH, eds. The Carbonic Anhydrases. Basel Switzerland, Birkhauser Verlag, 95104
Sterling D, Reithmeier RA, Casey JR (2001) Carbonic anhydrase: in the driver's seat for bicarbonate transport. JOP 2(suppl 4):165170[Medline]
Tajima Y, Okamura N, Sugita Y (1987) The activating effects of bicarbonate on sperm motility and respiration at ejaculation. Biochim Biophys Acta 924:519529[Medline]
Tripp BC, Smith K, Ferry JG (2001) Carbonic anhydrase: new insights for an ancient enzyme. J Biol Chem 276:4861548618
Tureci O, Sahin U, Vollmar E, Siemer S, Gottert E, Seitz G, Parkkila AK, et al. (1998) Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers. Proc Natl Acad Sci USA 95:76087613
Turner TT (1984) Resorption versus secretion in the rat epididymis. J Reprod Fertil 72:509514[Medline]
Turner TT (2002) Necessity's potion: inorganic ions and small organic molecules in the epididymal lumen. In Robaire B, Hinton BT, eds. The Epididymis: From Molecules to Clinical Practice. New York, Kluwer Academic/Plenum Publishers, 131150
Veri JP, Hermo L, Robaire B (1993) Immunocytochemical localization of the Yf subunit of glutathione S-transferase P shows regional variation in the staining of epithelial cells of the testis, efferent ducts, and epididymis of the male rat. J Androl 14:2344
Vince JW, Reithmeier RA (1998) Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte C1-/HCO3-exchanger. J Biol Chem 273:2843028437
Wong PY, Gong XD, Leung GP, Cheuk BLY (2002) Formation of the epididymal fluid microenvironment. In Robaire B, Hinton BT, eds. The Epididymis: From Molecules to Clinical Practice. New York, Kluwer Academic/Plenum Publishers, 119130
Zhou Q, Clarke L, Nie R, Carnes K, Lai LW, Lien YH, Verkman A, et al. (2001) Estrogen action and male fertility: roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function. Proc Natl Acad Sci USA 98:1413214137.