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Biology of Reproduction 61, 1521-1526 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Regional Expression and Androgen Regulation of Carbonic Anhydrase IV and IIin the Adult Rat Epididymis1

Kari Kaunisto2,a, Robert E. Flemingb, Joni Kneerb, William S. Slyc, and Hannu Rajaniemia

a Department of Anatomy and Cell Biology, University of Oulu, 90401 Oulu, Finland b Department of Pediatrics and c Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Carbonic anhydrase (CA) is implicated in the acidification of epididymal fluid and thereby in the regulation of sperm maturation and motility. Among the CA isoenzymes, CA IV and II have been shown to be present in the rat epididymal duct epithelium. In the present study, we examined the expression and androgen regulation of CA IV and II mRNAs along the epididymal duct. Northern blot analysis revealed the presence of CA II mRNA in all regions of the epididymis with the strongest signal in the corpus region, while CA IV mRNA was expressed predominantly in the corpus epididymidis. Three days after bilateral castration, CA IV and II mRNAs were decreased by 80–90% in the corpus epididymidis. Testosterone (T) replacement maintained the expression of CA mRNAs at 50–60% of the control levels, indicating that circulating androgens alone are not sufficient to recover the CA expression in the corpus region. However, unilateral castration did not affect the mRNA levels of CA IV and II, suggesting that factors in testicular fluid do not play a major role in the regulation of CA expression in the corpus epididymidis. Immunoblot analysis showed that CA IV protein levels decreased 3 days after castration, while T administration maintained the protein expression virtually at the precastration levels. These data demonstrate that mRNAs for CA IV and II are predominantly expressed in the corpus region of the rat epididymis and can be regulated by androgens in that region. The present data suggest that the regulation of CA expression in the corpus epididymidis by androgens contributes to the known androgen effects on epididymal acidification.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian epididymis provides an environment where the maturation and storage of sperm cells take place. During their passage through the epididymis, spermatozoa are exposed to the changing microenvironment provided by the epididymal fluid. The maintenance of the composition of luminal fluid is dependent on epithelial functions including transport of electrolytes and water across the luminal membrane [1] and protein absorption and secretion [2]. These functions are known to be dependent on the presence of androgens [3], which reach the epithelium from the vasculature as well as via the epididymal lumen [4].

The luminal fluid leaving the seminiferous tubules is acidified in the epididymal duct [5–7]. It has been proposed that this acidic milieu is involved in maintaining sperm cells in quiescence and in inhibiting acrosomal proteolytic enzymes during storage in the cauda epididymidis, in conjunction with factors including specific proteins and other ions [8, 9]. Micropuncture studies have shown that the acidification of epididymal fluid occurs mainly through absorption of bicarbonate ions [7]. Thus, epididymal acidification resembles that of the renal proximal tubule, where a Na+/H+ exchanger and a membrane-associated carbonic anhydrase, CA IV, in the luminal membrane are responsible for bicarbonate reabsorption [10]. Recently, we have demonstrated that the rat epididymal epithelium expresses CA IV and cytoplasmic CA II in segments where luminal fluid is acidified [11]. In addition, a population of cells rich in vacuolar-type H+-ATPase has been represented in the rat epididymis and vas deferens, suggesting that electrogenic proton secretion may also have a role in the acidification in the rat reproductive tract [12, 13].

Relatively little is known about the regulation of the epididymal acidification process. However, androgens have turned out to be important in maintaining acidification [14] as in other epithelial functions, such as protein synthesis and secretion [15, 16] and fluid absorption [17, 18]. The role of other hormones is unclear, although there is evidence that ion and water transport in the rat epididymis is responsive to aldosterone [19, 20].

We hypothesized that hormonal regulation of epididymal ion transport might be mediated via modulation of the expression of proteins involved in the transport process. In the present study, we investigated the expression and androgen regulation of CA mRNA in the rat epididymis in order to clarify the mechanism of hormonal influence in the acidification. We show here that the expression of CA IV and II mRNA and the CA IV protein in the rat corpus epididymidis is regulated by androgen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

Adult male rats of Sprague-Dawley strain (350–500 g) were purchased from the laboratory animal center of the University of Oulu. The rats were maintained under a 16L:8D photoperiod with free access to food and water. To determine the effect of circulating hormones on CA expression, the animals were assigned to the following three groups: intact rats, castrated rats (Cx), and Cx with testosterone (T). The castrations were performed transscrotally under fentanyl fluanisone-midazolam (Janssen Pharmaceutica, Beerse, Belgium) anesthesia. Immediately after castration the rats were implanted s.c. with slow-release pellets (Innovative Research of America, Sarasota, FL) releasing T (0.7 mg/day) or placebo. Unilateral castration studies were performed to determine whether testicular fluid-derived factor(s) in the epididymal fluid was required for the CA expression in the epididymis. The right testes were transscrotally removed under fentanyl fluanisone-midazolam anesthesia, while the epididymides were left intact. The left epididymides served as controls. The experimental design was approved by the Animal Experimentation Committee of the University of Oulu.

Tissue Isolation

Three days after the surgery, the rats were anesthetized with ether and killed by decapitation, and blood was collected to assay T. The epididymides were removed, dissected free from fat, and subdivided into four segments, i.e., initial segment, caput, corpus, and cauda, as previously described [11]. The tissues were immediately frozen in liquid nitrogen and stored at -80°C until use for RNA extraction or immunoblotting. Accessory sex gland (ventral prostate and seminal vesicles) weights were measured to monitor the hormonal effects on the experimental animals. To ensure rapid dissection of the tissues, the epididymides were not weighed. In addition, epididymal samples from each group and experiment were fixed in 4% paraformaldehyde overnight, dehydrated, and embedded in paraffin. Sections (5 µm) were cut and stained with hematoxylin and eosin for histological examination of epididymides from the experimental groups.

Serum T Assay

The blood was allowed to clot at 4°C, after which serum was collected by centrifugation at 4000 x g for 20 min at 4°C and stored at -80°C. T concentrations were measured by commercial RIA (generous gift from Orion Diagnostica, Espoo, Finland).

RNA Isolation and Analysis

Total RNA was isolated from frozen epididymal segments by RNA-Stat-60 (Tel-Test "B", Inc., Friendswood, TX), which utilizes a modification of the one-step procedure of Chomzynski and Sacchi [21]. RNA concentration and purity were determined by absorbance at 260 nm and 280 nm. Fifteen micrograms from each source was denatured in formaldehyde-containing buffer and electrophoresed in 1% agarose, 2.2 M formaldehyde gels. After electrophoresis, the gels were stained with ethidium bromide, and the size of the mRNA transcripts was estimated using RNA standards (Promega, Madison, WI). The RNA was transferred onto Nytran membranes (Schleicher & Schuell, Keene, NH) and immobilized by UV cross-linking. The blots were prehybridized in 50% formamide, 5-strength SSPE (saline-sodium phosphate EDTA), 5-strength Denhardt's solution, 50 mM NaPO4, pH 6.5, 200 µg/ml salmon sperm DNA, 1 mM EDTA, 0.1% SDS. Hybridization was performed using antisense RNA probes for rat CA IV [22] or rat CA II [23]. The probes were transcribed (Promega) from linearized plasmid templates and [32P]CTP (ICN Biochemical, Costa Mesa, CA). The blots were hybridized with the probes overnight at 65°C in the same solution and washed in double-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate)/0.2% SDS at 65°C for 20 min. After autoradiography and phosphorimagery, the blots were rehybridized with an antisense RNA probe for rat beta-actin (generously provided by K. O'Malley, Washington University, St. Louis, MO), the expression of which has been reported to be independent of androgens [24]. The blots were washed as above and reexposed to autoradiography and phosphorimagery. The autoradiographs were quantitated using phosphorimagery (ImageQuant, Molecular Dynamics, Sunnyvale, CA) or image analysis (Bio Image; Millipore, Ann Arbor, MI ). CA signal values were normalized to those obtained from beta-actin.

Immunoblotting

Tissue samples from epididymal segments were homogenized in 50 mM Tris-SO4 buffer (pH 7.0) containing protease inhibitors (1 mM PMSF, 1 mM O-phenantroline, 1 mM benzamidine). Protein concentrations were measured spectrophotometrically using a Bio-Rad (Richmond, CA) protein assay kit. Equal amounts of proteins (30 µg/lane) from total epididymal homogenates were dissolved in SDS sample buffer, separated by standard SDS-PAGE [25] using a 12% gel, and transferred onto nitrocellulose membranes (Schleicher & Schuell) according to standard method [26]. Parallel 12% gels were run and stained with Coomassie blue to confirm the equality of loading in each lane. The nitrocellulose strips were blocked with a solution containing 10% cow colostrum in 0.1% Tween 20/PBS (PBST) overnight, followed by incubation with anti-rat CA IV antiserum [27] at 1:3000 dilution in 1% BSA-PBST for 2 h. The nitrocellulose membranes were then washed with PBST and incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA) diluted 1:25 000 in BSA-PBST for 1 h. After washing in PBST, bound immunoglobulins were detected by enhanced chemiluminescence (ECL; Amersham, Little Chalfont, UK) according to the manufacturer's instructions.

Statistical Analysis

Data were analyzed using Student's t-test, and P values < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Longitudinal Distribution of CA IV and II mRNAs in the Rat Epididymis

Northern blot analysis for CA IV expression revealed a single band at 1.2 kilobases (kb) in corpus and caput regions of the rat epididymis and the rat colon, which served as a positive control (Fig. 1A). The size of the band corresponded to that earlier reported for rat CA IV [22]. The corpus epididymidis yielded the most intense signal level for CA IV mRNA, followed by the caput segment. In the initial segment and the cauda epididymidis, the CA IV mRNA was detected only after longer exposure times (data not shown). An antisense probe for CA II revealed a 1.7-kb band in all epididymal regions and the colon (Fig. 1B). The signal level for CA II mRNA was also distinctly highest in the corpus epididymidis, followed by the caput and cauda segments. The initial segment showed the lowest signal for CA II. The signal levels for both CA IV and II mRNA in the epididymis were clearly lower than in the rat colon.



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  		FIG. 1. Northern blot analysis of the regional distribution of CA IV and II mRNA in adult rat epididymis. Total RNA (15 µg) from the initial segment (IS), caput (CT), corpus (CO), and cauda (CD) epididymidis and proximal colon (PC) was subjected to Northern analysis and hybridized with 32P-labeled CA IV (A) and CA II (B) riboprobes. After stripping, the blots were reprobed with a radiolabeled beta-actin riboprobe (C)

Effects of Castration and Androgen Replacement

Three days after castration, significant decreases in the ventral prostate and seminal vesicle weights were observed in the rats implanted with placebo pellets as compared to the control animals (P < 0.05; Table 1). In the T group, the ventral prostate and seminal vesicle weights were, in turn, elevated compared to those in the controls (P < 0.05; Table 1). The serum T levels were significantly repressed in the bilaterally castrated rats as compared to the control rats and elevated in the Cx+T rats (P < 0.05; Table 1). The effects of different treatments on the epididymal histology were investigated by light microscopy using hematoxylin-eosin-stained paraffin sections. Epithelial cell height and the proportion of epithelial and stromal components were not markedly different among the three experimental groups (data not shown).


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  		TABLE 1. Serum T concentrations and organ weights (means ± SEM) of rats from different treatment groups

The androgen dependence of CA expression was studied in the corpus epididymidis. Three days after the bilateral castration, the steady state mRNA level for CA IV in the corpus epididymidis was 10% of that in the controls. T replacement maintained mRNA at 50% of the control levels, suggesting that circulating T is not the only factor regulating CA IV expression in this region (Fig. 2). The steady state mRNA levels for CA II were regulated in comparable manner. Bilateral castration for 3 days reduced the mRNA concentration to 20% of the control levels, while T administration maintained mRNA at 60% of the control levels (Fig. 2). Unilateral castration experiments were performed to determine the role of testicular fluid factors in the regulation of CA expression. Three days after unilateral castration, no apparent difference was seen in the expression of CA IV and CA II mRNAs in the corpus epididymidis between the castrated side and the intact side (data not shown).



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  		FIG. 2. Effects of bilateral castration and T replacement on steady state CA IV and CA II mRNA levels in corpus epididymidis. Total RNA was isolated from corpus epididymidis in the various treatment groups and probed with 32P-labeled antisense riboprobe against CA IV or CA II (A). The blots were then stripped and reprobed with a radiolabeled antisense riboprobe against beta-actin (B). The autoradiograms were quantitated by phosphorimager software or image analysis. The data are expressed as a percentage of the control value (mean ± SEM; n = 3) (C)

Immunoblot analysis was used to confirm the effect of androgen withdrawal and replacement on CA IV expression at the protein level in the corpus epididymidis. Immunoblots revealed a 39-kDa polypeptide band corresponding to the size of rat CA IV. There was a marked decrease in CA IV protein abundance 3 days after castration, whereas T treatment maintained the CA IV protein virtually at the control levels (Fig. 3). The experiment was repeated three times with comparable results.



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  		FIG. 3. Effects of bilateral castration and T replacement on CA IV protein expression in the corpus epididymidis. Total homogenates from corpus epididymidis of the control (C), castrated (Cx), and T-treated (Cx+T) rats were analyzed by immunoblot; 60 µg of protein was resolved per lane, and the blot was probed with anti-rat CA IV antibody and developed by ECL


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The importance of the acid-base microenvironment of epididymal fluid for sperm maturation and storage has been suggested by a number of studies on several species [9, 28, 29]. In particular, a lowered pH and/or bicarbonate concentration in epididymal fluid has been implicated in sperm quiescence during storage. Ion transport in the epididymis has been shown to be under hormonal control. Orchidectomy reduces and T administration restores the ion transport properties of the epididymis [18]. More recently, it has been shown that treatment of rats with estrogen or antiandrogen, flutamide, is associated with an impairment of epididymal fluid acidification [14, 30].

Several genes are expressed in a region-specific manner in the epididymis [31], suggesting region- and cell-specific differences in the functions of the epididymal duct. We have earlier shown by immunohistochemistry and immunoblotting that CA IV and II proteins are present in the rat epididymis, being located predominantly in the principal cells of the corpus epididymidis and, to a lesser extent, in some sporadic cells in other epididymal regions [11]. The results of the present Northern blot analysis are in complete accordance with these localization studies. The expression pattern of mRNAs indicated that CA IV and II are expressed predominantly in the corpus region. The low levels of CA II mRNA in the initial segment reflect the small amount of CA II-expressing narrow cells in that region [3].

The epididymis is a classical target tissue for androgens [3]. The main androgen responsible for maintaining epididymal structure and functions is 5{alpha}-dihydrotestosterone, which is produced through 5{alpha}-reduction of T in the epididymal cells [3]. Androgen receptors are present in the epididymal epithelium [32–34] and localized predominantly in principal cells [35, 36]. Bilateral castration and T replacement were used here to determine whether the steady state mRNA levels for CA IV and II are under androgen control. The study of the androgen dependence of CA expression was focused on the corpus region, since the mRNA levels for both CA isoenzymes in other regions are low. The results clearly suggest that mRNAs for both CA isoenzymes are under androgen control in the corpus region, since the mRNA levels decreased to 10–20% of the control levels 3 days after castration. The epithelium of the epididymis and the accessory sex glands begins to regress after castration [37, 38], thus making it possible that changes in mRNA expression after androgen withdrawal result from general involution of the epithelium and not from androgen-specific gene regulation. However, the duration of castration and hormone treatment in our study, 3 days, is relatively short for marked involution to occur [38]. In our study, this was confirmed by light microscopy of the tissue sections obtained from rats of all experimental groups. In addition, Fan and Robaire have shown recently that apoptotic cell death in the epithelium of corpus epididymidis, which is thought to be responsible for the involution, does not occur before Day 4 after castration [39].

Androgen replacement was not found to be sufficient to maintain precastration mRNA levels for CA IV and II in the corpus epididymidis, although the serum T concentrations obtained in the hormone treatment group distinctly exceeded the control levels. The incomplete recovery of CA mRNA levels by T replacement suggests a role for other factors in the regulation of CA expression. As the expression of several epididymal genes also appears to be regulated by factors derived from testicular fluid [40–43], unilateral castration experiments were carried out to investigate whether mRNA levels for CA IV and II are regulated by such factors. However, no significant difference in mRNA levels between the castrated side and the intact side was seen 3 days after unilateral castration, suggesting that testicular fluid factors may not play a major role in the regulation of CA expression in the corpus epididymidis. The possible role of other steroid hormones or direct effect of gonadotropins remains to be elucidated.

The effect of castration and androgen replacement on CA IV expression in the corpus epididymidis was also confirmed by immunoblotting. CA IV protein expression in the corpus region was shown to be under androgen control, albeit the observed effects were of smaller magnitude than with CA IV mRNA. CA II could not be blotted because its high content in red blood cells would have interfered with the interpretation of the results.

Several studies have indicated that androgen regulation of epididymal gene expression may be region specific [42–45]. For example, clusterin mRNA in the proximal epididymis is unaffected by castration and androgen administration but shows a distinct increase after castration in the corpus and cauda regions [44]. In the present study, androgen regulation of CA isoenzymes in regions other than the corpus may not be physiologically relevant because of their low expression. However, our preliminary studies have shown that CA IV mRNA in the cauda region seems to be up-regulated after castration, indicating that the regulation of CA IV expression also exhibits regional differences along the rat epididymis (unpublished results).

Steroid hormone regulation of CA II expression has been shown earlier in other tissues and cells. In the rat prostate, CA II is differentially regulated by androgens and estrogens [46, 47]. Moreover, CA II expression is increased in the human promyelocytic cell line, HL-60, and avian bone marrow cells by 1,25 dihydroxyvitamin D3 [48, 49] and in chicken erythroblasts by thyroid hormone [50]. The promoter region of the CA II gene has been shown to contain elements responsive to those steroids [49, 50], but the presence of androgen-responsive elements is not known. However, since only steady state levels of mRNA were examined in this study, the respective roles of the regulation of transcription, messenger stability, or posttranscriptional events remain to be solved. The present study is, to our knowledge, the first report suggesting that CA IV expression is also under hormonal regulation.

The results of the present study indicate that the mRNA expression of two key components of luminal acidification, CA IV and CA II, is almost exclusively restricted to the corpus epididymidis and is regulated by androgens in that region. The results suggest that the known influence of androgens on acidification may, at least in part, be mediated by the regulation of steady state mRNA levels for CA isoenzymes.


   ACKNOWLEDGMENTS
 
We thank Ms. Lissu Hukkanen and Mr. Eero Oja for expert technical assistance. We also thank Dr. Catherine Stolle and Dr. Karen O'Malley for providing cDNA clones for rat CA II and beta-actin.


   FOOTNOTES
 
1 This project was supported by Emil Aaltonen Foundation (to K.K.), Fleur de Lis Foundation (to R.F.), and NIH grants DK40163 and GM34182 (to W.S.S.). Back

2 Correspondence: Kari Kaunisto, Department of Anatomy and Cell Biology, University of Oulu, PL 5000, FIN-90401 Oulu, Finland. FAX: 358 8 537 5172; kari.kaunisto{at}oulu.fi Back

Accepted: July 22, 1999.

Received: April 21, 1999.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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