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Male Reproductive Tract |
Department of Zoology,3 University of Hong Kong, Pokfulam, Hong Kong
Department of Physiology,4 The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, male reproductive tract
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reverse transcription-polymerase chain reaction (RT-PCR) was employed to evaluate the presence of the secretin receptor and secretin messengers in the rat epididymis. Total RNA was isolated from the initial segment, caput, corpus, and cauda epididymidis of Sprague-Dawley rats weighing about 300 g using TRIzol reagent (Gibco BRL, Grand Island, NY). Two micrograms of the total RNA were used for the synthesis of the first-strand cDNA using oligo (dT)18 primer and Superscript II RNase H– reverse transcriptase (Superscript Preamplification System; Gibco BRL). Resulting first-strand cDNA was directly used for the PCR of secretin receptor and secretin.
Two pairs of specific primers were designed for the detection of secretin and its receptor in the epididymis according to the published cDNA sequences [13, 14]. The sense primer of secretin receptor was 5'-ACTGTGATGCCCATAAGGTG-3' and that of the antisense was 5'-AGGCGAAGACAATGTAGTGG-3,' which amplified specifically the coding region of the rat secretin receptor from 831 base pairs (bp) to 1290 bp and yielded a PCR product of 459 bp. The sense primer specific for secretin was 5'- GAC GTT CAC CAG CGA GCT CAG-3' and that of the antisense was 5'-CAC TCT GAA TGG TCG ACA GCA-3', which amplified the coding region of rat secretin from 131 bp to 400 bp and yielded a PCR product of 270 bp. S-16 was used as the internal standard [15]. The sense primer of S-16 was 5'-TCC GCT GCA CTC CGT TCA AGT CTT-3' and that of the antisense was 5'-GCC AAA CTT CTT CTT GGA TTC GCA GCG- 3', which amplified the coding region from 15 bp to 400 bp and yielded a PCR product of 385 bp. The PCR were carried out by combining the following reagents in a final volume of 50 µl: 1x PCR buffer, 1.5 mmol/ L MgCl2, 5 mmol/L dNTP, 10 µmol/L primer pair, 1 U Taq DNA polymerase, and 2 µl cDNA template. These PCR mixtures were subjected to 25–28 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The PCR products were resolved on a 1.5% agarose gel and visualized under ultraviolet light following ethidium bromide staining. The rat jejunum and pancreas were used as positive control for secretin and secretin receptor, respectively.
Western Blotting of Secretin and Its Receptor
Protein expression of secretin and its receptor were examined by Western blotting. In brief, isolated epididymal epithelial cells were sonicated at 4°C in 5 mM sodium phosphate (Na2PO4) buffer and centrifuged at 2500 x g for 15 min. The protein content of the supernatant was determined spectrophotometrically using a commercial bicinchoninic acid assay (Sigma-Aldrich Co., St. Louis, MO). Fifty micrograms of protein were resolved on 10% or 15% (w/v) SDS-polyacrylamide gel and electrotransferred to nitrocellulose membranes. The membranes were subsequently blocked with 5% (w/v) nonfat dry milk in PBS overnight at 4°C, followed by incubation with rabbit anti-secretin (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-secretin receptor antibody (1:1000 [v/v] dilution in blocking solution). Visualization of secretin and its receptor proteins in rat epididymal protein extracts was achieved by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) according to manufacturer's manual. Jejunum and pancreas protein extract were used as the positive control.
Culture of Epithelia from Rat Cauda Epididymidis
All experiments on animals were performed in accordance with guidelines on the use of laboratory animals established by the Animal Ethics Committee of the Chinese University of Hong Kong. The procedures of tissue culture have been described previously [16, 17]. Sprague-Dawley rats weighing about 300 g were used as a source of cauda epididymidis. The rats were killed by CO2 inhalation. The lower abdomen was opened and the caudal part was separated from the rest of the epididymis. The tissue was then finely cut with scissors and placed in sterile Hanks balanced salt solution (HBSS) containing 0.25% (w/v) trypsin. Tissue was incubated for 30 min at 32°C with vigorous shaking (150 strokes/min) for 30 min. The tissue was separated by low-speed centrifugation (800 x g, 5 min). The supernatent was discarded and the pellet was resuspended in HBSS containing 0.1% (w/v) collagenase for 60 min at 32°C with vigorous shaking. Cells were separated by centrifugation at 800 x g for 5 min. The pellet was resuspended in Eagle minimum essential medium containing nonessential amino acids (0.1 mM), sodium pyruvate (1 mM), glutamine (4 mM), 5
-dihydrotestoterone (1 nM), 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml). The cell suspension was incubated for 4 h at 32°C in 5% CO2. During this period, fibroblasts and smooth muscle cells attached to the bottom of the culture flask while the epididymal epithelial cells remained suspended. The cell suspension was decanted and seeded into the wells of Millipore filter assemblies with a diameter of 0.4 cm2 (cell concentration 105 cells/ml, plating density 5 x 104 cells/cm2 filter) floating on 15 ml of culture medium. Cultures were incubated for 3 days at 32°C in 5% CO2. Thereafter, the monolayers reached confluency and were ready for the measurement of short-circuit current.
Short-Circuit Current Measurement
Confluent epididymal monolayers were clamped between two halves of Ussing chambers with a 0.6 cm2 window. The tissue was short-circuited by the use of a voltage-clamp amplifier (DVC 1000; World Precision Instrument, New Haven, CT). The short-circuit current (Isc) was displayed on a pen recorder. Transepithelial resistance was obtained from Ohm law by clamping the tissue intermittently at a voltage at 0.1–0.3 mV displaced from zero. The two channels of the amplifier were mostly used simultaneously on parallel monolayers so that studies could be made under control and experimental conditions. In most situations, the monolayers were bathed on both sides with Krebs-Henseleit (K-H) solution containing (in mM) NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.8; KH2PO4, 1.8; NaHCO3, 25; and glucose, 1.4. This solution had a pH of 7.3–7.4 when bubbled with 95% O2, 5% CO2. In some experiments, a Cl–-free or HCO3–-free K-H solution was used. The former was made by substitution of sodium gluconate, potassium gluconate, and CaSO4 for NaCl, KCl, and CaCl2, respectively. In the latter, HCO3– was replaced by 25 mM Hepes and the solution was bubbled with pure O2 to maintain pH at 7.4. Secretin and PACAP were obtained from Bachem Bioscience Inc. (King of Prussia, PA) and were reconstituted in distilled water and kept below –20°C before use. Piroxicam, Cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride (MDL-12330A), bumetanide, and acetazolamide were from Sigma, Co. (St. Louis, MO); lysylbradykinin (LBK) was from Cambridge Research Biochemicals (Cambridge, UK).
Immunohistochemical Detection of Secretinin the Epididymis
Adult, male Sprague-Dawley rats weighing about 300 g were used. Tissues were fixed in Bouin solution at room temperature overnight. The fixed tissues were dehydrated, embedded in paraffin, and sectioned at 5 µm in thickness. Paraffin sections were heat treated to 60°C, dewaxed, and hydrated in PBS. Antigen retrieval was performed by treatment in 10 mM citrate buffer (pH 6) for 2 min in a microwave oven. Slides were then treated with methanol containing 3% (v/v) hydrogen peroxide for 30 min to quench the endogenous peroxidase activity. After rinsing with PBS, sections were incubated in normal blocking serum (Vectastain Elite ABC kit, Vector PK-6101; Vector Laboratories, Burlingame, CA) for 1 h. Afterwards, excess serum was drained off and then the slides were incubated with rabbit anti-secretin (mouse) serum (1:200) (Santa Cruz Biotechnology) or rabbit anti-secretin receptor (mouse) serum (1:600) (anti-serum of secretin receptor was raised using a synthetic peptide [R-A-E-C-L-R-E-L- S-E-E-K-K] that is present in the mouse secretin receptor) diluted in PBS at 4°C overnight. Sections were rinsed with PBS and incubated with biotinylated secondary antibody. After washing with PBS, the sections were incubated with Vectastain Elite ABC reagent (ABC kit) for 30 min and finally washed with PBS. Visualization was achieved by immersing sections in peroxidase substrate solution (DAB substrate kit; Vector Laboratories) until desired stain intensity developed. Slides were rinsed with nanopure water, counterstained with Lillie-Mayer hematoxylin, dehydrated, followed by xylene and mounted for observation. Negative controls were obtained by the omission of primary antibodies.
Statistical Analysis
Comparisons between groups of data were made by Student t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our laboratory has recently located secretin and secretin receptor transcripts in distinct neuronal cell populations [2, 18], and this finding prompted us to investigate the expression of these mRNAs in other tissues. Reverse transcription-polymerase chain reaction was used to detect the expression of secretin and its receptor in the male reproductive tract. The rat jejunum and pancreas were used as positive controls for secretin [19] and secretin receptor [20], respectively. PCR products of secretin (270 bp) and secretin receptor (459 bp) were amplified by RT-PCR from RNAs extracted from the rat initial segment, caput, corpus, and cauda epididymidis. The highest expression of secretin mRNA is seen in the initial segment. The expression level decreases through the caput and corpus epididymidis, reaching an undetectable level in the cauda (Fig. 1A). Similarly, expression of the secretin receptor mRNA was most prominent in the initial segment and the caput, corpus and cauda epididymidis shared a similar but lower level of expression (Fig. 1B). Western blots revealed that both secretin and secretin receptor are expressed by the epididymis (Fig. 1C).
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Effect of Secretin on Short-Circuit Current
When incubated in normal Krebs-Henseleit solution, epididymal epithelia exhibited a potential difference (PD) of 2–4 mV, a short-circuit current (Isc) of 1–2 µA cm–2 and a transepithelial resistance of about 500 
cm–2 [16]. Addition of secretin to the basolateral side caused a dose-dependent increase in the Isc (Fig. 2). The current increased gradually, reaching a peak level after 3 min, and maintained at that level thereafter. A maximal response (
Isc, 2.98 + 0.16 µA cm–2, n = 6) was obtained with 100 nM secretin. The EC50 value (concentration producing 50% maximal response) was 50 nM. Addition to the apical side produced a smaller response (maximal response 1.51 µA cm–2, n = 6). The EC50 value was 50 nM (Fig. 2, inset).
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Effects of Chloride or Bicarbonate Removal and Inhibitors of Chloride and Bicarbonate Transport
The short-circuit current response to secretin was studied in the absence of extracellular Cl– or CO2-bicarbonate. When epithelia were incubated in K-H solution containing Cl–, the Isc response to secretin (100 nM, basolateral application) was 2.9 8 + 0.16 µA cm–2 (n = 6). In epithelia incubated in Cl–-free solution, Isc was 1.25 + 0.08 µA cm–2 (n = 6). The difference is significant statistically (P < 0.01, Student unpaired t-test). In epithelia incubated in HCO3–-free solution, Isc was 1.82 + 0.14 µA cm–2 (n = 6; significantly different from control at P < 0.01). In Cl- and bicarbonate-free solution, Isc was 0.23 + 0.05 µA cm–2 (n = 6, significantly different from control at P < 0.001) (Fig. 3).
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In some experiments, epithelia were stimulated with secretin (100 nM, basolateral application). When the Isc has reached a plateau, bumetanide, inhibitor of the Na/K/2Cl symport, and acetazolamide, inhibitor of carbonic anhydrase, were added to the basolateral side of the epithelium in succession. It was found that bumetanide and acetazolamide inhibited 72% and 28% of the secretin-stimulated current, respectively, irrespective of the order in which the two drugs were added (Fig. 3). These results indicate that chloride and bicarbonate secretion accounted for the secretin-stimulated Isc.
Effects of COX and Adenylate Cyclase Inhibitors
Previous work has shown that angiotensin [21], bradykinin [17], and endothelin [22] stimulate chloride secretion by increasing formation of prostaglandins. Experiments were performed on epithelia pretreated with piroxicam, an inhibitor of prostaglandin synthesis, before stimulation with secretin, PACAP, or bradykinin. Figure 4 shows that pretreatment with piroxicam blocked the Isc response to PACAP added apically (significantly different from control at P < 0.01) and bradykinin added basolaterally (significantly different from the control at P < 0.01) but was without effect on secretin added either apically or basolaterally.
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In the pancreatic duct cells, secretin stimulates HCO3– secretion through an increase in intracellular cAMP [23]. An adenylate cyclase inhibitor, MDL-12330A [24], was used to inhibit formation of intracellular cAMP. Figure 5 shows pretreatment with MDL-12330A almost completely suppressed the Isc response to secretin (100 nM).
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Immunolocalization of Secretin and Secretin Receptorin the Rat Epididymis
Positive immunoreactivity for secretin was found in the apical cytoplasm of the principal cells of the initial segment and the caput epididymis (Fig. 6). The principal cells of the caput region were more heavily stained than those of the initial segment. The corpus and cauda epididymidis were stained negative.
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Secretin receptor-like immunoreactivity was more diffuse throughout the epithelium of the caput as well as the cauda epididymidis (Fig. 7). Occasional darker bands could be seen associated with the apical and basal border of the principal cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the epididymis, fluid secretion is influenced by neurotransmitters and humoral agents produced by and acting on the epithelium. The epididymis is therefore functionally similar to the gastrointestinal tract. The gut hormone secretin was the first hormone discovered in 1902 by Bayliss and Starling [1]. It is released by the duodenum and jejunum and acts on the pancreatic ductal cells to stimulate secretion of a fluid rich in sodium bicarbonate. Like other members of the secretin/glucagon/VIP superfamily, secretin is also found in the brain, where it may regulate or modulate central nervous activities [2, 18]. In view of the resemblance between the epithelia in the epididymis and exocrine pancreas, it is of interest to know if secretin plays a role in controlling electrolyte transport in the epididymis. This study reports on the expression and localization of secretin and its receptor in the epididymis and provides the first evidence that secretin may serve as an autocrine/paracrine factor that is involved in the formation of the luminal fluid by controlling chloride/fluid secretion by the principal cells.
The stimulation of Isc by secretin is due to the concurrent electrogenic chloride and bicarbonate secretion, as removal of chloride or CO2-bicarbonate caused a partial reduction of the Isc. Furthermore, bumetanide, which inhibits chloride secretion through inhibition of the Na/K/2Cl symport, and acetazolamide, which inhibits bicarbonate secretion through inhibition of the enzyme carbonic anhydrase, produced comparable reductions in the current to that seen after chloride or CO2-bicarbonate removal. The effects of bumetanide and acetazolamide are additive as a combination of the two agents completely abrogated the Isc response to secretin (Fig. 3, lower panel). These results show that, as with other peptide hormones viz., angiotensin [21], bradykinin [17], CGRP [32], endothelin [22], and PACAP [7], secretin regulates anion and fluid transport in the epididymis.
Previous work in our laboratory has shown PACAP, a member of the secretin/VIP superfamily, can stimulate chloride secretion in epithelia cultured from the rat epididymis [7]. However, the effects of secretin and PACAP appear quite distinct on the following counts: first, PACAP works only from the apical side of the epithelium to stimulate chloride secretion and not bicarbonate secretion; basolateral application is without effect [7]. Second, the stimulation of chloride secretion by PACAP requires local prostaglandin synthesis as piroxicam pretreatment abolished this response (Fig. 4). This is in stark contrast with secretin, which acts on the apical as well as the basolateral side of the epithelium to stimulate chloride and bicarbonate secretion. These effects are not affected by piroxicam pretreatment and hence are prostaglandin independent (Fig. 4).
Semiquantitative RT-PCR detected secretin mRNA and secretin receptor mRNA in the epididymis. The secretin gene is expressed most abundantly in the proximal part of the epididymis, with activity decreasing toward the cauda region (Fig. 1A). This pattern of regional distribution is matched by immunohistochemical studies, which show high secretin-like imunoreactivity in the initial segment and caput epididymidis but much less in the corpus and almost none in cauda epididymidis (Fig. 6). It is of interest to note the secretin-like immunoreactivity is localized in the supranuclear areas toward the apical pole of the epithelium. This phenomenon is consistent with the local synthesis and packaging of secretin peptide ready for release by the principal cells. The secretin receptor, on the other hand, is present in the proximal as well as the distal part of the epididymis. The protein is present in the principal cells in a more diffuse manner, with little preponderance in the clear cells (Fig. 7). Bands of more intense immunoreactivity, however, can be seen in the apical and basal borders of the principal cells throughout the length of the epididymis. This pattern of distribution of secretin and its receptor is consistent with the notion that secretin is synthesized by the proximal part (initial segment and caput) of the epididymis and is secreted into the lumen as well as into the peritubular blood. The luminal secretin molecules then act in an autocrine manner on the secretin receptors present on the apical membrane of the principal cells that secrete them, or are carried downstream where they act on the apical secretin receptors in the cauda epididymis in a paracrine fashion. Secretin molecules that are secreted peritubularly into the circulation act on the secretin receptors located in the basolateral domain of the principal cells of the entire epididymis. Through these G-protein-coupled receptors, secretin increases formation of cAMP, which stimulates secretion of chloride and bicarbonate to create an osmotic driving force for water secretion. The physiological findings that secretin stimulates chloride and bicarbonate secretion when added to the apical or basolateral side of cultured epididymal epithelia lend functional support to this contention. The present work has therefore shown that secretin, which was originally identified as a gut peptide a century ago and more recently proposed as a neuropeptide [2, 18], may have a role to play in the epididymis. It is of interest to note that the effects of secretin on electrolyte transport in the exocrine pancreas and the epididymis are similar, suggesting that secretin may play a more global role in regulating bicarbonate/Cl– fluxes in fluid-secreting epithelia. This hypothesis awaits further studies in the future.
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FOOTNOTES |
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2 Correspondence: Professor P.Y.D. Wong, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong. FAX: 852 2603 5022; patrickwong{at}cuhk.edu.hk
Received: 12 October 2003.
First decision: 28 October 2003.
Accepted: 22 January 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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