Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fisher, J. S.
Right arrow Articles by Breton, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fisher, J. S.
Right arrow Articles by Breton, S.
Agricola
Right arrow Articles by Fisher, J. S.
Right arrow Articles by Breton, S.
Biology of Reproduction 67, 1106-1114 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Modulation of the Onset of Postnatal Development of H+-ATPase-Rich Cells by Steroid Hormones in Rat Epididymis1

Jane S. Fisher2,a, Nuria Pastor-Solerb, Richard M. Sharpea, and Sylvie Bretonb,c

a Medical Research Council, Human Reproductive Sciences Unit, Edinburgh EH16 4SB, United Kingdom b Renal Unit and Program in Membrane Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129 c Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vacuolar type H+-ATPase is involved in lumenal acidification of the epididymis. This protein is highly expressed in narrow and clear cells where it is located in the apical pole, and it contributes to proton secretion into the lumen. We have previously shown that in rats, epididymal cells rich in H+ATPase appear during postnatal development and reach maximal numbers at 3–4 wk of age. The factors that regulate the appearance of these cells have not been investigated, but androgens, estrogens, or both may be involved. This study examined whether neonatal administration of estrogens (diethylstilbestrol [DES] or ethinyl estradiol) or an antiandrogen (flutamide), or the suppression of androgen production via administration of a GnRH antagonist (GnRHa), was able to alter the appearance of cells rich in H+-ATPase in the rat epididymis when assessed at age 25 days. Surprisingly, all of these treatments were able to significantly reduce the number of H+-ATPase positive cells; this was determined by immunofluorescence and confirmed by Western blotting. In contrast, neonatal coadministration of DES and testosterone maintained the expression of H+-ATPase in the epididymis at Day 25 despite the high level of concomitant estrogen exposure. These findings indicate that androgens, acting via the androgen receptor, are essential for the normal development of epididymal cells rich in H+-ATPase, and that treatments that interfere directly or indirectly with androgen production (GnRHa, DES) or action (flutamide, DES) will result in reduced expression of H+-ATPase. Our findings do not exclude the possibility that estrogens can directly suppress the postnatal development of cells in the epididymis that are rich in H+-ATPase, but if this is the case, this suppression can be prevented by testosterone administration.

epididymis, estradiol, male reproductive tract, steroid hormones, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The major function of the epididymis is to maintain a lumenal fluid environment, which is optimal for the maturation and, in many animals, the storage of spermatozoa [1]. The ability of epididymal epithelial cells to alter the ionic composition of the lumenal fluid has been measured, some of the major transport proteins involved in this process have been identified, and their sites of expression have been characterized [1–7]. Transepithelial transport of water and various anions and solutes take place in the epididymis [3]. Results from our laboratory indicate that vacuolar-type H+-ATPase is a key player in the establishment of an acidic pH in the epididymal lumen. In vitro studies on isolated rat vas deferens using inhibitors of proton secretion, such as bafilomycin, suggested that H+-ATPase is a major source of acidification [8]. Epididymal cells that contain H+-ATPase also coexpress carbonic anhydrase type 2 (CAII) [9, 10], suggesting that bicarbonate transport proteins are also important in lumenal acidification. The involvement of bicarbonate transport in the acidification process was confirmed in isolated vas deferens [11]. Epididymal cells rich in H+-ATPase share similar features to type A intercalated cells of the kidney collecting duct, which also express vacuolar-type H+-ATPase and CAII, and they play a major role in urinary acidification [12]. These proton-secreting cells are often referred to as "mitochondria-rich cells" [13, 14].

Previous studies have localized H+-ATPase to specific epithelial cell types in all regions of the epididymis; these are apical (or narrow) cells within the initial segment region, and clear (or light) cells within the caput, corpus, and cauda regions of the epididymis [7, 8]. The timing of postnatal differentiation of narrow and clear cells has been assessed primarily by electron microscopy, and they are fully differentiated around the time of sexual maturation (~42 days) in the rat [15]. Our further studies on postnatal expression of H+-ATPase in the rat epididymis have shown that cells rich in H+-ATPase are not present immediately after birth and that at Postnatal Day 14, cells that express H+-ATPase are still rare in the epididymis, although they are readily detected in the vas deferens [10]. H+-ATPase reaches its peak level of expression in the epididymis at 3–4 wk after birth [10]. The factors that regulate the postnatal maturation of the cell types that express H+-ATPase within the epididymis are not fully understood.

It is well known that testosterone is required in order to stabilize the Wolffian duct prior to its differentiation into the epididymis, vas deferens, and seminal vesicles [16, 17]. Testosterone exerts its effects via activation of the androgen receptor (AR), as does the 5{alpha}-reduced androgen, dihydrotestosterone (DHT). It is less clear whether the metabolites of testosterone, namely, estradiol or the major metabolite of DHT, 5{alpha}-androstane-3{alpha}, 17ß-diol, have any role to play in epididymal differentiation or maturation. Steroid receptors for both androgens and estrogens are present in the epididymis during postnatal development [18–21], and we have established that neonatal manipulation of these hormones in the rat can result in major changes in the structural differentiation of the epididymis and vas deferens during the peripubertal period [21]; estrogens or the androgen:estrogen balance appear to be the most important factors in this [20]. These findings raise the question of whether such structural changes are associated with altered expression of epididymal proteins such as H+-ATPase. The present study, therefore, assessed whether neonatal hormonal manipulation alters the subsequent development of cells rich in H+-ATPase during pubertal development. Manipulation of the levels and action of estrogen, androgen, or both were performed neonatally during the period of onset of the expression of H+-ATPase. The consequences of these treatments on the development of H+-ATPase-rich cells were determined in the rat epididymis at Postnatal Day 25, when the number of these cells should be at its peak [10].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals, Treatments, Sample Collection, and Processing

Wistar rats were bred and maintained in the Medical Research Council animal facility (Edinburgh). All animal studies were performed under license from and according to the legal requirements of the United Kingdom Home Office. Rats were administered one of the following treatments postnatally. In our studies the day of birth was assigned as Day 1. Based on the nomenclature below, the rats treated in a preliminary study (study 1) received either treatment 1, 2, 3, or 5. In a follow-up study (study 2) a cohort of rats was treated with one of the six regimens listed below.

1. Subcutaneous injection of 20 µl of corn oil administered on alternate days from Day 2 through Day 12 (control);
2. Subcutaneous injection of ethinyl estradiol (Sigma Chemical Company, Dorset, U.K.) at a dose of 10 µg/20 µl of corn oil administered on alternate days from Day 2 through Day 12;
3. Subcutaneous injection of DES (Sigma) at a dose of 10 µg/20 µl of corn oil administered on alternate days from Day 2 through Day 12;
4. Subcutaneous injection of 10 µg of DES coadministered with 200 µg of testosterone esters (DES + TE; Sustanon, Organon Labs, Cambridge, U.K.) administered in 20 µl of corn oil on alternate days from Day 2 through Day 12. The rationale for the TE dose has been fully described elsewhere [20];
5. Subcutaneous injection of 10 mg/kg of a long-acting gonadotropin-releasing hormone antagonist (GnRHa; Antarelix, Europeptides, Argenteuil, France) in 5% mannitol administered on Postnatal Days 2 and 5. This regimen has been shown to abolish gonadotropin secretion until Postnatal Days 15–20 [22] and to lower testosterone levels [23];
6. Subcutaneous injection of the androgen-receptor antagonist, flutamide (Sigma), administered at a dose of 50 mg/kg in 20 µl of corn oil on alternate days from Day 2 through Day 12. These animals were compared with an additional group of control rats.

For all treatments, pups were weaned at 22 days and fed standard rat and mouse breeding diet no. 3 (SDS, Dundee, Scotland). Animals were killed on Day 25 by inhalation of CO2, followed by cervical dislocation. The right epididymis from each animal was fixed for ~5 h in periodate-lysine paraformaldehyde (PLP), which contains 2% paraformaldehyde [24], and the whole left epididymis was snap-frozen and stored at -70°C prior to protein extraction. In study 2, the right testis was weighed after necropsy and the other reproductive organs were snap-frozen for use in other experiments.

Testosterone Assay

Plasma testosterone levels were measured using an ELISA adapted from an earlier radioimmunoassay method [25] as detailed elsewhere [26]. The limit of detection was ~12 pg/ml.

Immunocytochemistry

The PLP-fixed epididymides of control and treated 25-day-old rats were cryoprotected by immersion in 30% sucrose in PBS for at least 4 h. The entire epididymis (head, body, and tail) was then mounted in OTC compound Tissue-Tek (Miles Inc., Elkhart, IN) and frozen at -29°C in a Reichert Frigocut cryostat (Reichert Jung, Derry, NH). Sections were cut at 4 µm, picked up onto Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA), and kept at 4°C.

For immunostaining, the sections were hydrated for 10 min in PBS, and antigen retrieval was performed by incubating the slides for 4 min with 1% SDS in PBS. After three washes in PBS for 5 min each, nonspecific staining was blocked by applying to the slides a solution of 1% BSA in PBS/0.02% sodium azide for 15 min. An affinity-purified rabbit primary antibody against the 56-kDa subunit of the vacuolar H+-ATPase was applied to the slides at a concentration of 1:1000 in DAKO diluent (DAKO Corporation, Carpinteria, CA) for 90 min at room temperature. Sections were then washed twice for 5 min each time in PBS containing 2.7% NaCl to reduce nonspecific binding, followed by one wash in normal PBS. For immunofluorescence labeling, a secondary goat anti-rabbit antibody coupled to the fluorophore CY3 (Jackson Immunologicals, West Grove, PA) was applied to the slides at a 1:800 dilution in DAKO diluent for 1 h at room temperature, followed by the same series of washes that were used for the primary antibody. The slides were mounted in Vectashield diluted 1:1 with Tris buffer pH 8.5 (Vector Laboratories, Burlingame, CA).

Control incubations were performed using antibodies that had been preabsorbed with an excess of the immunizing peptide (11 amino acids that correspond to the C-terminus of the 56 kDa, B1 subunit of the H+-ATPase) prior to the first incubation step.

Quantification of H+-ATPase Immunoexpression

Study 1 Black-and-white photographs were taken of the whole epididymis of each animal. The number of H+-ATPase positive cells on each photograph was counted and the basement membrane of the associated tubule was drawn around using a Wacom graphics tablet (Vancouver, WA). The mean number of immunopositive cells per millimeter of epididymal tubular basal membrane was calculated for each animal.

Study 2 Sections were examined using a Nikon Eclipse 800 epifluorescence microscope (Micro Video Instruments Inc., Avon, MA). Black-and-white images were obtained using a Hamamatsu Orca CCD digital camera (Micro Video Instruments Inc.) and IP lab Spectrum software (Scanalytics, Vienna, VA). The images were taken using a 20x objective.

Cells with positive fluorescence staining for vacuolar H+-ATPase in the epididymides were counted on digital images while they appeared on the computer screen. The basal perimeter of the epididymal tubules that were analyzed was measured using IP lab Spectrum software. The final number of positive cells per millimeter in the perimeter of the basal membrane of the tubule was calculated for each image in Microsoft Excel. The number of rats analyzed in each cohort is listed in Table 1.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Testis weight and number of rats per cohort in study 2 at age 25 days in vehicle-treated (control) rats and in animals treated neonatally with GnRHa, DES, EE, DES + TE, or in a separate experiment, flutamide

Protein Extraction

Whole epididymides from each treatment group were powdered in a porcelain mortar with a pestle under liquid nitrogen. The powder was scraped into an Eppendorf tube and stored on dry ice. Protein was extracted by the addition of 200 µl of cold extraction buffer (10 mM Hepes pH 7.9, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM PMSF, and 1x Complete protease inhibitor cocktail [Roche, Lewes, U.K.]). The tissue remained on ice for 15 min before adding 25 µl of 10% Nonident P-40 (Sigma). The tube was then vortexed three times for 10 sec. The tube was then centrifuged at 12 000 x g for 1 min at 4°C, the supernatant was decanted, and 100-µg aliquots were frozen on dry ice before being stored at -40°C.

Western Blot Analysis

The expression of H+-ATPase and ß-actin protein were determined in 25-day-old rat epididymal protein extracts using Western blotting. Protein markers (Bio-Rad Laboratories, U.K.) and protein samples (from all treatment groups) were separated by SDS-PAGE using gradient gels (4%–20%; Novex precast gels, Invitrogen,). The gels were run at 110 V for ~2 h before blotting onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Watford, U.K.) at 33 V for 180 min using Tris/Gly transfer buffer (Invitrogen). The membranes were blocked for 1 h in 5% milk/TBS, 50 mM Tris-HCl, and 150 mM NaCl. The membranes were washed thoroughly in TBST (TBS containing 0.05% Tween-20; Sigma) before the primary antibody was added. An affinity-purified chicken antiserum against the 31-kDa subunit of the H+-ATPase was added at a dilution of 1:1000 (TBS containing 0.02% sodium azide) and incubated at 4°C overnight. Alternatively, a commercially available ß-actin monoclonal antiserum (Sigma) was added to the membrane at a dilution of 1:8000 and incubated overnight. The membranes were washed at least four times for 15 min in TBST. The second antibody was then added to the membrane and incubated for 1 h (for H+-ATPase, we used a rabbit anti-chicken immunoglobulin G conjugated to horseradish peroxidase [Sigma]; for ß-actin, we used a goat anti-mouse immunoglobulin G conjugated to peroxidase) before being thoroughly washed in TBST. The H+-ATPase protein was detected using enhanced chemiluminescence (Amersham, Buckinghamshire, U.K.) according to the manufacturer's instructions. The membranes were then exposed to Hyperfilm-LS (Kodak) until optimal development of the signal was detected. The film was scanned (Snapscan-e-40; AGFA, Middlesex, U.K.) using Scanwise software (AGFA). The image was then mounted in Photoshop 5.5 (Adobe, San Jose, CA).

Statistical Analysis

Data are expressed as means ± SEM. One-way ANOVA was performed followed by the Tukey test. Statistical significance was conducted at a level of confidence of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The results of study 1 are presented first, followed by those obtained in study 2. Study 1 examined the effect of neonatal treatment with DES, ethinyl estradiol (EE), or GnRHa on the number of cells in the epididymis that were rich with H+-ATPase at 25 days of age. The number of cells positive for H+-ATPase was then quantified and calculated as the number of positive cells per millimeter of epididymal tubular basal membrane.

Study 1

Representative images illustrating the number of H+-ATPase-rich cells in the cauda epididymis of control and treated animals are shown in Figure 1. Panels A and B illustrate H+-ATPase immunostaining within control epididymides. Both images demonstrate that H+-ATPase is principally expressed in the apical pole of a subpopulation of epithelial cells. In control animals, a large number of H+-ATPase-positive cells were detected at Postnatal Day 25. However, after neonatal administration of either GnRHa, DES (Fig. 1, C and D, respectively), or EE (data not shown), the number of cells expressing H+-ATPase was much smaller. These results were confirmed by quantifying the number of H+-ATPase immunopositive cells for each animal. Epididymides from control animals had a mean of 27 ± 4.5 H+-ATPase positive cells per millimeter, whereas treatment with either DES or EE significantly reduced (P < 0.001) this to 2.44 ± 0.92 and 5.86 ± 4.7, respectively. Neonatal treatment with GnRHa also reduced the number of H+-ATPase positive cells per millimeter (14.71 ± 7.7),but this decrease failed to reach statistical significance. This group had three animals, two of which showed a major decrease, and a third that showed little difference from control values. This variation in GnRHa response was one reason why a larger follow-up study was performed.



View larger version (140K):
[in this window]
[in a new window]
  		FIG. 1. Immunoexpression of H+-ATPase in the 25-day rat epididymis visualized by fluorescence (bright green staining). Tissue sections from a control rat cauda epididymidis are shown at low (A) and high power magnification (B) to demonstrate the location of H+-ATPase in the apical membrane. Comparable low power sections from rats treated neonatally with GnRHa (C), or DES (D) are shown to demonstrate the reduced frequency of immunopositive cells. Bars = 40 µm in A, C, and D, and 20 µm in B

Study 2

The treatments performed in study 1 were repeated and, in addition, a cohort of rats was also treated neonatally with DES + TE to determine whether the administration of testosterone could prevent the negative effects of DES observed in study 1. Similarly, to determine whether androgen action via the AR is important in establishing or maintaining cells that are rich in H+-ATPase, a cohort of rats was treated neonatally with the AR antagonist flutamide.

Testis Weight

Administration of GnRHa, EE, DES, or DES + TE induced similar effects on testis weight at 25 days of age, causing >85% reduction in weight (Table 1). These findings demonstrate the effectiveness of the respective treatments in retarding testicular development [20]. In contrast, neonatal administration of flutamide was without significant effect on testicular weight in this particular study.

Specificity of the H+-ATPase Antibody

To confirm specificity of the H+-ATPase antibody, immunofluorescence experiments were conducted using antibodies that had been preabsorbed with an excess of the immunizing peptide prior to the first incubation step. As shown in Figure 2, the immunoreactivity was completely abolished by preabsorption of the antibodies (compare Fig. 2, A and B), indicating specificity of the staining.



View larger version (101K):
[in this window]
[in a new window]
  		FIG. 2. Specificity of the H+-ATPase antibody. Epididymis sections were incubated with the H+-ATPase antibody (A) or with antibody that had been preabsorbed with the immunizing peptide (B). The staining is completely abolished in the presence of the peptide

Plasma Testosterone Levels in Neonatally Treated Rats

Testosterone levels in 25-day-old rats were significantly reduced in all cohorts treated neonatally with GnRHa, DES, or EE as shown in Figure 3A. Plasma testosterone levels were assessed in 18-day-old rats after neonatal flutamide treatment (Fig. 3B). This graph illustrates the variable effect of flutamide treatment on testosterone levels, but overall, there was no significant increase in levels after flutamide treatment.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 3. The effect of neonatal treatment with vehicle, GnRHa (n = 8), DES (n = 8), or EE (n = 7) on plasma testosterone levels (ng/ml) at Day 25 (A) and the effect of neonatal treatment with flutamide (n = 5) or vehicle (n = 5) on plasma testosterone levels at Day 18 (B). ***P < 0.001, in comparison with the respective control group. Data are means ± SEM

Quantification of H+-ATPase-Rich Cells in Control and Neonatally Treated Rats

The epididymides of control rats all exhibited a large subpopulation of H+-ATPase immunopositive cells. As demonstrated in study 1, the immunofluorescence was largely present in the apical pole (Fig. 4A). The morphology of the positive cells appeared similar throughout the epididymis, with tall columnar epithelial cells being evident in all regions. Panels B and C in Figure 4 illustrate cauda epididymides from Day 25 rats after neonatal treatment with GnRHa or DES, respectively (EE not shown), and demonstrate broadly similar reductions in the number of H+-ATPase-positive cells to those illustrated in Figure 1. The epididymides of animals treated with GnRHa and DES showed retarded development, as evidenced by smaller epididymal lumens when compared with control rats or those treated with DES + TE (Fig. 4, A and D, respectively). Coadministration of DES + TE resulted in the maintenance of epididymal epithelial H+-ATPase immunoexpression at levels similar to controls. Morphologically, the epididymal epithelium of the animals treated with DES + TE appeared broadly similar to that in controls. However, despite the maintenance of H+-ATPase levels, the testes weight of these animals were similar to those measured in rats treated with DES alone or with GnRHa (Table 1). The number of H+-ATPase-positive cells per millimeter of basal membrane was quantified in each group (Fig. 5). Rats treated neonatally with either GnRHa or DES exhibited ~65% fewer H+-ATPase positive cells, whereas treatment with EE caused a reduction of 45% (Fig. 5). Animals coadministered DES + TE neonatally had H+-ATPase positive cells that were comparable in number to the control cohort at Day 25 (50.3 ± 13.8 vs. 49.6 ± 9.8 immunopositive cells per millimeter of epididymal basal membrane, respectively).



View larger version (107K):
[in this window]
[in a new window]
  		FIG. 4. Immunoexpression of H+-ATPase in Day 25 rat cauda epididymidis from a control rat (A) and rats treated neonatally with either GnRHa (B), DES (C), or DES + TE (D). Note the reduction in H+-ATPase immunostaining similar to that shown in Figure 1 after DES and GnRHa treatment, but also note the similarity in the intensity and number of immunopositive cells in control animals and those treated with DES + TE. Bar = 40 µm



View larger version (64K):
[in this window]
[in a new window]
  		FIG. 5. Quantification of the number of H+-ATPase-positive cells per millimeter of epididymal tubule basal membrane at Day 25 in control rats and in rats treated neonatally with either GnRHa, DES, EE, or DES + TE. **P < 0.01, ***P < 0.001 compared with control. Data are means ± SEM for n = 4–8 (as stated in Table 1)

A separate cohort of rats was treated with vehicle or the androgen receptor antagonist flutamide (Fig. 6, A and B, respectively). Similar to GnRHa, DES, and EE treatments, administration of flutamide induced a reduction in the number of H+-ATPase positive cells, although this effect was less marked than in animals treated with DES alone or with GnRHa. The tubule lumen was smaller in the treated group, indicating that flutamide treatment also retarded epididymal lumen development. These observations are in accordance with published data [20]. Quantification analysis showed that the rats treated neonatally with flutamide exhibited a 34% reduction in the mean number of H+-ATPase positive cells per millimeter of epididymal basal membrane (Fig. 7) compared with their controls.



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 6. Immunoexpression of H+-ATPase in Day 25 rat cauda epididymis from a control rat (A) and from an animal treated neonatally with flutamide (B). Note the clear reduction in the number of H+-ATPase-rich cells in the latter group. Bars = 40 µm



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 7. Quantification of the number of H+-ATPase-positive cells per millimeter of epididymal tubule basal membrane at Day 25 in control rats and in rats treated neonatally with flutamide. **P < 0.01 in comparison with control. Data are means ± SEM for n = 3

Western Blot Analysis of H+-ATPase Protein Levels

To confirm the changes in the expression of H+-ATPase that was detected by immunofluorescence after neonatal hormone treatments, Western blot analysis was performed (Fig. 8). In homogenates from control epididymides, the affinity-purified antibody revealed a strong band at around 31 kDa and an additional band at around 60 kDa, as we have described previously [27] (Fig. 8, A and B). Treatments with GnRHa, DES, or EE induced a marked reduction in the intensity of these bands, whereas coadministration of DES + TE prevented the reduction in H+-ATPase expression that occurred after DES treatment alone (Fig. 8A). Similarly, Western blot analysis confirmed that neonatal treatment with flutamide induced a reduction in H+-ATPase protein levels in comparison to control levels (Fig. 8B). To demonstrate equal protein loading in each sample, a Western blot against ß-actin was also performed (Fig. 8, C and D).



View larger version (74K):
[in this window]
[in a new window]
  		FIG. 8. A and B) Western blot analysis of H+-ATPase protein levels in epididymides from 25-day control rats (lanes 1 and 6) and from rats treated neonatally with GnRHa (lane 2), DES (lane 3), EE (lane 4), DES + TE (lane 5), or flutamide (lane 7; this lane should be compared with the control in lane 6). A significant decrease in the H+-ATPase signal was observed after GnRHa, DES, EE, and flutamide treatments. Coadministration of TE with DES prevented the inhibition induced by DES treatment alone. C and D) Western blot analysis of ß-actin protein levels in the same groups. No difference in signal was observed, suggesting that equal protein levels were added to each lane


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Vacuolar H+-ATPase is involved in the process of lumenal acidification in the male reproductive tract of both rats and humans [7, 8, 12, 27]. The acidic lumenal environment is believed to play a role in maintaining and storing sperm in an immotile state prior to ejaculation [17, 28, 29]. We have previously shown that H+-ATPase is expressed in a subpopulation of epididymal epithelial cells, the narrow cells in the initial segments, and clear cells in the remainder of the epididymis [7, 8, 10, 12], and that these cells are present as early as 2 wk after birth [10]. At this time point, a large number of H+ATPase-rich cells were observed in the vas deferens, whereas only a few positive cells were detected in the epididymis, suggesting that the ability to secrete protons may begin in the vas deferens and progress back toward the epididymis during postnatal development. The peak time of H+-ATPase expression in the epididymis occurs at around Postnatal Day 25 [10]. Therefore, both the onset and peak expression of the H+-ATPase occur at ages when the testicular production of testosterone is relatively low [28, 30], suggesting that the expression of H+-ATPase might be regulated by factors other than androgens, or in addition to them. Alternatively, the low levels of androgens present in prenatal and prepubertal male rats might be sufficient to trigger the development of H+-ATPase-rich cells. In addition, the epididymis acquires detectable activity of the enzyme {Delta}4-5{alpha}-reductase [30], which converts testosterone to the more potent DHT at 21 days after birth, and it remains possible that significant levels of local DHT might be present and contribute to the initiation of the H+ATPase-rich cell phenotype during early postnatal development.

Previous data in the literature suggested that narrow and clear cells (also called apical mitochondria-rich cells) were under the control of estrogens and not androgens [14]. Another study showed that lumenal acidification in the rat epididymis is under androgen control [31]. The current study was, therefore, designed to test whether neonatal manipulation of sex steroid levels affected the development of epididymal H+-ATPase-rich cells.

Our data suggest that administration of estrogen (DES or EE), GnRHa, or flutamide can suppress the postnatal development of H+-ATPase-rich cells. We also observed a significant reduction in testis weights and in plasma testosterone levels in all treatment groups, except for animals treated with flutamide (DES + TE was not assessed for plasma testosterone levels). These treatments (GnRHa, DES, EE, and DES + TE) probably retarded testicular growth via suppression of the hypothalamo-pituitary axis and subsequent suppression of gonadotropin secretion, because we have shown that all of these treatments significantly reduce FSH levels ([22, 32, 33] and unpublished data). This is supported by the similarity in the degree of testis weight reduction observed between GnRHa and DES/EE treatments, as has also been reported in other studies [20, 34], and by the observation that coadministration of GnRHa and DES has no greater effect on testis weight than either treatment alone (unpublished data). The comparable levels of the reduction in the number of H+-ATPase-rich cells observed in animals treated with DES and GnRHa are suggestive of a common mechanism in altering the development of this cell phenotype. This most likely centers on interference with androgen production, action, or both, because both GnRHa and DES treatments suppress blood levels of testosterone [23], and DES (but not GnRHa) treatment causes a major reduction in expression of the androgen receptor protein in the epididymis and vas deferens [20, 35]. The possibility that the DES effects observed in the present study were at least partially attributed to lower levels of androgens was confirmed by the observation that neonatal coadministration of testosterone with DES was able to restore epididymal expression of H+-ATPase. In addition, H+-ATPase expression in the epididymis was reduced by neonatal flutamide administration. Because flutamide induced no change in testis weight or plasma testosterone levels, indicating no suppression of the hypothalamo-pituitary axis, the reduction in the number of H+ATPase-rich cells by this agent is presumably due to direct inhibition of epididymal androgen action. Thus, the most unifying explanation for our findings is that altered expression of H+-ATPase in the Day 25 epididymis results directly from insufficient androgen action during neonatal development. However, our results do not exclude the possibility that the effects observed in animals treated neonatally with estrogens, GnRHa, or flutamide are secondary to a general retardation of development of the epididymis, as indicated by the immature morphological appearance of the epididymis under these treatments, which has been described in other studies [20].

We have shown previously that some other morphological effects on the reproductive tract that were observed after neonatal DES administration, such as rete testis distension, loss of aquaporin-1 expression in the efferent ducts, and malformation of the epididymis/vas deferens, were not altered after GnRHa administration [36], suggesting an estrogenic action of DES in addition to androgen suppression [20]. In addition, previous reports have suggested that estrogens might be involved in the maturation of some epididymal cell types that express H+-ATPase [10, 14]. Epididymal epithelial cells are structurally undifferentiated in all regions of the epididymis at Postnatal Day 21 [15], and some H+-ATPase expressing cell types (i.e., caput and corpus epididymal clear cells) are not fully differentiated until Day 49 despite being exposed to high testosterone levels, which is consistent with a role for other factors in their maturation. Given that the postnatal epididymis contains receptors for estrogen receptor ß (ERß) [21], and that the major circulating steroid during postnatal development is 3{alpha}-diol [37, 38], a putative ligand for ERß, an argument can be proposed in favor of a role for estrogens in the development of H+ATPase-rich cells.

Recent evidence suggests that 3{alpha}-diol is a ligand for ERs (preferentially ERß) and that it acts to regulate AR levels in the rat prostate [39]. Whether or not 3{alpha}-diol regulates AR levels in any other regions of the male reproductive tract has not been investigated, but because one of the major effects of neonatal DES administration is the virtual abolition of AR protein expression in the epididymis [20], the potential involvement of 3{alpha}-diol is a possibility. Studies of the male rat anterior pituitary have shown that the conversion of DHT to 3{alpha}-diol is reversible and that 3{alpha}-hydroxysteroid dehydrogenase (3{alpha}-HSD) can convert 3{alpha}-diol back into DHT [40]. 3{alpha}-HSD is expressed in neonatal epididymides and may catalyze the local conversion of 3{alpha}-diol from the circulation into DHT, thus promoting the development of H+-ATPase-rich cells. It is possible that this metabolite is capable of acting as a potent androgen via conversion to DHT by 3{alpha}-HSD or as an estrogenic ligand with ER{alpha} or ERß [39]. Because 3{alpha}-HSD, ARs, and ERs are all present in the pubertal epididymis, it is not possible to determine whether 3{alpha}-diol acts in one or all of these roles. The potential dual role of this metabolite reinforces how intimately the actions of androgens and estrogens may be linked. The data presented in our study demonstrate that DES can reduce H+-ATPase expression but to the same level as neonatal treatment with GnRHa. So, whereas our findings do not exclude direct estrogenic effects on the epididymis, they imply that this role is secondary to, and can be overcome by androgens. The change in development of cells rich in H+-ATPase induced by neonatal administration of steroids emphasizes the importance of the steroid hormone environment for maintaining the normal timing and intensity of expression of genes such as H+-ATPase and that altering the steroid environment can repress or retard their expression.

In summary, this study examined whether androgens, estrogens, or both are involved in the development of cells that are rich in H+-ATPase. Our data show that treatment with either a potent GnRHa or estrogen (DES or EE) is sufficient to significantly reduce the expression of this protein. It is possible that these compounds induce this reduction via suppressing the hypothalamo-pituitary axis with consequent reduction in androgen levels [41, 42] and subsequent loss of androgen receptor protein expression or action in the reproductive tract [20, 35]. Because cells rich in H+-ATPase were not lost in animals treated neonatally with DES + TE, this suggests that androgens are capable of preventing some of the effects induced by DES. This role for androgens is supported by the reduction in the number of H+ATPase-rich cells observed in rats treated with the androgen receptor antagonist flutamide during postnatal development. Additional support is provided by the previous observation that flutamide treatment of the adult rat epididymis induces an increase in lumenal pH, consistent with androgen action being responsible for maintaining a low pH within the epididymis [31]. Our data suggest that normal serum testosterone levels and functional epididymal ARs are required for normal developmental expression of H+-ATPase-rich cells in the epididymis.


   ACKNOWLEDGMENTS
 
The authors thank Dennis Brown for his assistance throughout this study and for his constructive criticism during preparation of the manuscript. Thanks are also due to Jim MacDonald and Dennis Doogan for help with animal husbandry.


   FOOTNOTES
 
1 This work was supported by grant QLK4-1999-01422 from the European Union to J.F. and by grants HD40793 and DK38452 from the National Institutes of Health (NIH) to S.B. N.P.-S. was supported by NIH NRSA award HD08684. Back

2 Correspondence: Jane Fisher, Medical Research Council, Human Reproductive Sciences Unit, 49 Little France Crescent, Old Dalkeith Rd., Edinburgh EH16 4SB, U.K. FAX: 44 0 131 228 5571; j.fisher{at}hrsu.mrc.ac.uk Back

Received: 9 November 2001.

First decision: 27 November 2001.

Accepted: 2 May 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hinton B, Palladino M. Epididymal epithelium: its contribution to the formation of a luminal fluid microenvironment. Microsc Res Tech 1995 30:67-81[CrossRef][Medline]
  2. Ilio KY, Hess RA. Structure and function of the ductuli efferentes. Microsc Res Tech 1994 29:432-467[CrossRef][Medline]
  3. Levine N, Marsh DJ. 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 1971 213:557-570[Abstract/Free Full Text]
  4. Leung A, Wong P. Studies of transepithelial Cl- transport in cultured cauda epididymal cells of rats by the short-circuit current method. J Physiol (Lond) 1992 457:391-406[Abstract/Free Full Text]
  5. Bagnis C, Marsolais M, Biemesderfer D, Laprade R, Breton S. Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis. Am J Physiol Renal Physiol 2001 208:F426-F436
  6. Jensen LJ, Stuart-Tilley AK, Peters LL, Lux SE, Alper SL, Breton S. Immunolocalization of AE2 anion exchanger in rat and mouse epididymis. Biol Reprod 1999 61:973-980[Abstract/Free Full Text]
  7. Brown D, Lui B, Gluck S, Sabolic I. A plasma membrane proton ATPase in specialized cells of rat epididymis. Am J Physiol 1992 263:C913-C916[Abstract/Free Full Text]
  8. Breton S, Smith P, Lui B, Brown D. Acidification of the male reproductive tract by a proton pumping (H+)ATPase. Nat Med 1996 2:470-472[CrossRef][Medline]
  9. Kaunisto K, Parkkila S, Parkkila AK, Waheed A, Sly WS, Rajaniemi H. Expression of carbonic anhydrase isoenzymes IV and II in rat epididymal duct. Biol Reprod 1995 52:1350-1357[Abstract]
  10. Breton S, Tyszkowski R, Sabolic I, Brown D. Postnatal development of H+ATPase (proton-pump)-rich cells in rat epididymis. Histochem Cell Biol 1999 111:97-105[CrossRef][Medline]
  11. Breton S, Hammar K, Smith PJ, Brown D. Proton secretion in the male reproductive tract: involvement of Cl-independent HCO-3 transport. Am J Physiol 1998 257:C1134-C1142
  12. Brown D, Breton S. H+-ATPase-dependent lumenal acidification in the kidney collecting duct and the epididymis/vas deferens: vesicle recycling and transcytotic pathways. J Exp Biol 2000 203:137-145[Abstract]
  13. Brown D, Breton S. Mitochondria-rich, proton secreting epithelial cells. J Exp Biol 1996 199:2345-2358[Abstract]
  14. Martinez G, Regadera J, Cobo P, Palacois J, Paniagua R, Nistal M. The apical mitochondria-rich cells of the mammalian epididymis. Andrologia 1995 27:195-206[Medline]
  15. Hermo L, Barin K, Robaire B. Structural differentiation of the epithelial cells of the testicular excurrent duct system during postnatal development. Anat Rec 1992 233:205-228[CrossRef][Medline]
  16. Orgebin-Crist MC, Danzo BJ, Davis JT. Endocrine control of the development and maintenance of sperm fertilizing ability in the epididymis. In: Greep RO, Astwood E (eds.), Handbook of Physiology, vol. 5. Washington: American Physiological Society; 1975: 319–338
  17. Robaire B, Hermo L. Efferent ducts, epididymis, and vas deferens: structure, functions and their regulation. In: Knobil E, Neill J (eds.), The Physiology of Reproduction, vol. 2. New York: Raven Press; 1988: 999–1080
  18. Saunders PTK, Maguire SM, Gaughan J, Millar MR. Expression of oestrogen receptor beta (ERß) in multiple rat tissues visualised by immunocytochemistry. J Endocrinol 1997 154:R13-R16[Abstract/Free Full Text]
  19. Saunders P, Fisher J, Sharpe R, Millar M. Expression of oestrogen receptor beta (ERß) occurs in multiple cell types, including some germ cells, in the rat testis. J Endocrinol 1998 156:R13-R17[Abstract]
  20. McKinnell C, Atanassova N, Williams K, Fisher J, Walker M, Turner K, Saunders PTK, Sharpe R. Suppression of androgen action and the induction of gross abnormalities of the reproductive tract in male rats treated neonatally with diethylstilbestrol. J Androl 2001 22:323-338[Abstract]
  21. Atanassova N, McKinnell C, Williams K, Turner KJ, Fisher J, Saunders PTK, Millar M, Sharpe R. Age-, cell- and region-specific immunoexpression of ER{alpha} (but not ERß) during postnatal development of the epididymis and vas deferens of the rat and disruption of this pattern by neonatal treatment with diethystilboestrol. Endocrinology 2001 142:874-886[Abstract/Free Full Text]
  22. Sharpe R, Turner K, McKinnell C, Groome N, Atanassova N, Millar M, Buchanan D, Cooke P. Inhibin B levels in plasma of the male rat from birth to adulthood: effect of experimental manipulation of Sertoli cell number. J Androl 1999 20:94-101[Abstract/Free Full Text]
  23. Williams K, McKinnell C, Saunders PTK, Walker M, Fisher J, Turner KJ, Atanassova N, Sharpe R. Neonatal exposure to potent and environmental oestrogens and abnormalities of the male reproductive system in the rat: evidence for the importance of the androgen-oestrogen balance and assessment of the relevance to man. Hum Reprod Update 2001 7:236-247[Abstract/Free Full Text]
  24. McLean IW, Nakane PK. Periodate-lysine paraformaldehyde. A new fixative for immunoelectron microscopy. J Histochem 1974 22:1077-1083[Abstract]
  25. Corker C, Davidson D. Radioimmunoassay of testosterone in various biological fluids without chromatography. J Steroid Biochem 1978 9:373-374[CrossRef][Medline]
  26. Atanassova N, McKinnell C, Walker M, Turner K, Fisher J, Morley M, Millar M, Groome N, Sharpe R. Permanent effects of neonatal estrogen exposure in rats on reproductive hormone levels, Sertoli cell number, and the efficiency of spermatogenesis in adulthood. Endocrinology 1999 140:5364-5373[Abstract/Free Full Text]
  27. Herak-Kramberger CM, Breton S, Brown D, Kraus O, Sabolic I. Distribution of the vacuolar H+ ATPase along the rat and human male reproductive tract. Biol Reprod 2001 64:1699-1707[Abstract/Free Full Text]
  28. Robaire B, Viger RS. Regulation of epididymal epithelial cell functions. Biol Reprod 1995 52:226-236[Abstract]
  29. Jones RC, Murdoch RN. Regulation of the motility and metabolism of spermatozoa for storage in the epididymis of eutherian and marsupial mammals. Reprod Fertil Dev 1996 8:553-568[CrossRef][Medline]
  30. Scheer H, Robaire B. Steroid 5{alpha}-reductase and 3{alpha}-hydroxysteroid dehydrogenase in the rat epididymis during development. Endocrinology 1980 107:948-953[Abstract/Free Full Text]
  31. Calflisch C. Effect of a nonsteroidal antiandrogen, flutamide on intralumenal acidification in rat testis and epididymis. Andrologia 1993 25:363-367[Medline]
  32. Sharpe R, Atanassova N, McKinnell C. Abnormalities in functional development of the Sertoli cells in rats treated neonatally with diethlystilbestrol: a possible role for estrogens in Sertoli cell development. Biol Reprod 1998 59:1084-1094[Abstract/Free Full Text]
  33. Atanassova N, McKinnell C, Turner KJ, Walker M, Fisher JS, Morley M, Millar M, Groome NP, Sharpe RM. Comparative effects of neonatal exposure of male rats to potent and weak (environmental) estrogens on spermatogenesis at puberty and the relationship to adult testis size and fertility: evidence for stimulatory effects of low levels. Endocrinology 2000 141:3898-3907[Abstract/Free Full Text]
  34. Sharpe R, Atanassova N, McKinnel C, Parte P, Turner K, Fisher J, Kerr J, Groome N, Macpherson S, Millar M, Saunders P. Abnormalities in functional development of the Sertoli cell in rats treated neonatally with diethylstilbestrol: a possible role for estrogens in Sertoli cell development. Biol Reprod 1998 59:1084-1094
  35. Sharpe R, McKinnell C, Atanassova N, Williams K, Turner KJ, Saunders PTK, Walker M, Millar M, Fisher J. Interactions of androgens and oestrogens in development of the testis and male reproductive tract. In: Jegou B, Pineau C, Saez JM (eds.), Testis, Epididymis and Technologies in the Year 2000. Berlin, Heidelberg: Springer-Verlag; 2000: 173–195
  36. Fisher JS, Turner KJ, Fraser HM, Saunders PTK, Brown D, Sharpe RM. 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 1998 139:3935-3945[Abstract/Free Full Text]
  37. Moger WH. Serum 5{alpha}-androstane-3{alpha},17ß-diol, androsterone, and testosterone concentrations in the male rat. Influence of age and gonadotropin stimulation. Endocrinology 1977 100:1027-1032[Abstract/Free Full Text]
  38. Moger W, Murphy P. Serum 5{alpha}-androstane-3{alpha},17ß-diol, androsterone, and testosterone concentrations in the immature male rat: influence of time of day. J Endocrinol 1977 75:177-178[Abstract/Free Full Text]
  39. Weihua Z, Makela S, Andersson L, Salmi S, Webster J, Jensen E, Nilsson S, Warner M, Gustafsson J. A role for estrogen receptor ß in the regulation of growth of the ventral prostate. Proc Natl Acad Sci U S A 2001 98:6330-6335[Abstract/Free Full Text]
  40. Pilven A, Thieulant M, Ducouret B, Samperez S, Jouan P. Rapid and intensive conversion of 5alpha-androstane-3alpha, 17beta-diol into 5alpha-dihydrotestosterone in the male rat anterior pituitary: in vivo and in vitro studies. Steroids 1976 28:349-358[CrossRef][Medline]
  41. Brown-Grant K, Fink G, Greig F, Murray MA. Altered sexual development in male rats after oestrogen administration during the neonatal period. J Reprod Fertil 1975 44:25-42[Abstract/Free Full Text]
  42. Bellido C, Pinilla L, Aguilar R, Gaytan F, Aguilar E. Possible role of changes in postnatal gonadotrophin concentrations on permanent impairment of the reproductive system in neonatally oestrogenized male rats. J Reprod Fertil 1990 90:369-374[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
G. I. Gorodeski, U. Hopfer, C. C. Liu, and E. Margles
Estrogen Acidifies Vaginal pH by Up-Regulation of Proton Secretion via the Apical Membrane of Vaginal-Ectocervical Epithelial Cells
Endocrinology, February 1, 2005; 146(2): 816 - 824.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Pastor-Soler, V. Beaulieu, T. N. Litvin, N. Da Silva, Y. Chen, D. Brown, J. Buck, L. R. Levin, and S. Breton
Bicarbonate-regulated Adenylyl Cyclase (sAC) Is a Sensor That Regulates pH-dependent V-ATPase Recycling
J. Biol. Chem., December 5, 2003; 278(49): 49523 - 49529.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fisher, J. S.
Right arrow Articles by Breton, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fisher, J. S.
Right arrow Articles by Breton, S.
Agricola
Right arrow Articles by Fisher, J. S.
Right arrow Articles by Breton, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS