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Endocrinology, doi:10.1210/en.2004-0711
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*OXYTOCIN
Endocrinology Vol. 145, No. 12 5767-5773
Copyright © 2004 by The Endocrine Society

Regulation of 5{alpha}-Reductase Isoforms by Oxytocin in the Rat Ventral Prostate

S. J. Assinder, C. Johnson, K. King and H. D. Nicholson

Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, Dunedin, New Zealand

Address all correspondence and requests for reprints to: Dr. Stephen Assinder, Andrology Research Group of Otago, Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, P.O. Box 913, Dunedin, New Zealand. E-mail: stephen.assinder{at}stonebow.otago.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Oxytocin (OT) is present in the male reproductive tract, where it is known to modulate contractility, cell growth, and steroidogenesis. Little is known about how OT regulates these processes. This study describes the localization of OT receptor in the rat ventral prostate and investigates if OT regulates gene expression and/or activity of 5{alpha}-reductase isoforms I and II. The ventral prostates of adult male Wistar rats were collected following daily sc administration of saline (control), OT, a specific OT antagonist or both OT plus antagonist for 3 d. Expression of the OT receptor was identified in the ventral prostate by RT-PCR and Western blot, and confirmed to be a single active binding site by radioreceptor assay. Immunohistochemistry localized the receptor to the epithelium of prostatic acini and to the stromal tissue. Real-time RT-PCR determined that OT treatment significantly reduced expression of 5{alpha}-reductase I but significantly increased 5{alpha}-reductase II expression in the ventral prostate. Activity of both isoforms of 5{alpha}-reductase was significantly increased by OT, resulting in increased concentration of prostatic dihydrotestosterone. In conclusion, OT is involved in regulating conversion of testosterone to the biologically active dihydrotestosterone in the rat ventral prostate. It does so by differential regulation of 5{alpha}-reductase isoforms I and II.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE MALE, OXYTOCIN (OT) is released into the circulation from the posterior pituitary and is also produced locally within the reproductive tissues (1, 2). In the testis (3, 4) and epididymis (5), OT has been implicated in the regulation of contractility. OT has similar effects in both the human and rat prostate, producing an increase in prostatic tone and contractile activity (6).

As well as affecting contractility, OT modulates testosterone production in the rat, both in vitro (7) and in vivo (8). Perhaps more significantly, OT also promotes the conversion of testosterone to dihydrotestosterone (DHT) by stimulating activity of the enzyme 5{alpha}-reductase (9, 10). Two forms of 5{alpha}-reductase are present in the prostate (11, 12). In the rat prostate, 5{alpha}-reductase type I is expressed by basal epithelial cells, whereas type II isoform is predominantly found in stromal tissue (12). The prostate is dependent on DHT, and regulation of this steroid is important for normal growth and function. At present, it is not known if both isoforms are affected by OT or if the actions of OT are restricted to just one of the isoforms. Furthermore, it is not known if OT acts to increase activity of existing enzymes and/or increases expression of 5{alpha}-reductase isoforms.

A prerequisite for OT’s actions in the prostate is the presence of a specific OT receptor. OT receptor has been identified in the prostates of both monkey and human (13, 14). However, in the rat, where most of the biological effects of the peptide have been studied, the presence and localization of receptors in the prostate have not been described.

The aims of this study were to: 1) determine the presence and localization of OT receptor in the rat; 2) determine whether exposure to OT increases activity of both 5{alpha}-reductase isoforms; and 3) investigate whether gene expression of 5{alpha}-reductase isoforms is affected by OT in the rat prostate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissues
This study was approved by the Otago University Animal Ethics Committee. Adult male Wistar rats (250–300 g) were housed under a controlled temperature and light regime with food and water provided ad libitum. After euthanasia by CO2 inhalation, the ventral prostate was removed and either fixed in Bouin’s fluid for immunocytochemistry or snap frozen in liquid nitrogen and stored at minus 70 C for subsequent protein and mRNA analyses.

Detection of OT receptor mRNA
Total RNA was extracted from frozen samples with Trizol (Invitrogen Life Technologies, Gaithersburg, MD) according to the manufacturer’s protocol. Eighty nanograms of total RNA were used to produce cDNA with Superscript RT system (Invitrogen Life Technologies, Carlsbad, CA) from random hexamers according to the manufacturer’s instructions. A total of 1 µl of the reverse transcription reaction mix was used in PCRs. A hot start PCR protocol was used to amplify cDNA, employing Amplitaq gold DNA polymerase (Applied Biosystems, Piscataway, NJ). OT receptor cDNA was amplified from 5'-CCAAGGAAGCCTCGGCCTTCATC-3' and 5'-GATGGCTGGGAGCAGCTCCTCTG-3' primers (1 cycle of 94 C for 2 min, 35 cycles of 94 C for 30 sec, 65 C for 30 sec, and 72 C for 1 min) to generate a predicted 247-bp amplicon corresponding to nucleotides 911-1158 of the rat OT receptor cDNA (15). The predicted amplicon spanned a 12-kb intron boundary so as to avoid the amplification of contaminating genomic DNA and was confirmed by the absence of PCR product in control reactions that used non-reverse-transcribed total RNA as template. All PCR products were analyzed by electrophoresis in 1% (wt/vol) agarose gel containing 0.1 µg·ml–1 ethidium bromide and viewed under UV transillumination. Identity of products generated were determined by sequence analysis of amplicons generated from two separate rat prostate samples. These were isolated from the agarose gel using Ultrafree-DA centrifugal units (Millipore Corp., Bedford, MA) and subcloned into pGem-T easy vector plasmid (Promega, Madison, WI) according to the manufacturer’s instructions. The fragment sequence was determined by the dideoxy chain-termination method using the SL1FRH primer to initiate the reaction.

Detection of OT receptor protein
The presence of OT receptor protein was confirmed using a Western blot procedure as previously described (16). Crude tissue extracts of rat prostates were separated according to size by discontinuous SDS-PAGE (15%, 24:1 bis:acrylamide gels) and electrotransferred onto polyvinylidene difluoride membrane. Blots were probed with anti-OT receptor antibody 020 (16) at a 1:2000 dilution. Detection of bound antibody was achieved by chemiluminescence [BM chemiluminescence (POD), Roche Molecular Biochemicals UK, Lewes, East Sussex, UK] and exposure of Hyperfilm (Amersham International, Amersham, Buckinghamshire, UK). An extract of rat uterus was included as a positive control tissue, and liver extract was included as a negative control tissue. Control blots of all samples were incubated with normal rabbit serum in place of the primary antibody.

Localization of the OT receptor in the rat ventral prostate
A high-temperature antigen retrieval technique as described by Whittington et al. (16) was employed. Control sections were incubated with preimmune serum in place of the primary antibody, or with antiserum 020, diluted as above but preincubated with immunogen (16). Rat uterus was included as a positive control tissue.

Radioligand receptor binding assay
Ventral prostates, testes (positive control) and skeletal muscle (negative control) from adult rats (n = 6) were homogenized in 10 vol of 10 mmol·liter–1 Tris-HCl (pH 7.4) containing 1 mmol·liter–1 EDTA, 0.01% (wt/vol) Bacitracin, and 0.02% (wt/vol) soy bean trypsin inhibitor. Tissue homogenates were centrifuged at 1000 x g for 10 min at 4 C, the supernatant collected and recentrifuged at 60,000 x g for 30 min at 4 C. Pellets were resuspended in homogenization buffer and sonicated (10–14 Hz) for 5 sec. The suspension was recentrifuged at 60,000 x g for 30 min at 4 C. The pellet was resuspended and protein concentration determined. Saturation binding assays were performed to assess the total and nonspecific binding of OT antagonist (OTA) d(CH2)5[Tyr(Me)2, Thr4,Tyr-NH29]OVT (Bachem, Essex, UK) radiolabeled with 125I by the chloramine-T method (18). Total binding was measured by incubating homogenates (100 µg of protein) with 125I-OTA (0.01–1 nmol·liter–1) in manganese buffer [20 mmol·liter–1 Tris-HCl (pH 7.4) containing 15 mmol·liter–1 MnCl2, 0.6 mmol·liter–1 EDTA, 0.1% (wt/vol) BSA, 0.006% (wt/vol) bacitracin, and 0.0012% (wt/vol) soy bean trypsin inhibitor] for 1 h at 25 C. Nonspecific binding was assessed by coincubating homogenates with either 10 µmol·liter–1 OT or 2 µmol·liter–1 OTA. After incubation, tubes were rapidly filtered through GF-BASGF/B filters (1 µm pore diameter; Whatman, Brentford, Middlesex, UK) using a 24-place cell harvester (Brandell Corp., Montreal, Quebec, Canada) and radioactivity of filters counted. Receptor concentrations (maximum binding capacity) and affinity [dissociation constant (Kd)] were determined from nonlinear regression rectangular hyperbola (binding isotherms) of specific binding (fmol·mg–1 protein) vs. the radioligand concentration.

Effects of OT treatment on expression of 5{alpha}-reductase I and 5{alpha}-reductase II in the rat prostate
Four groups of adult Wistar rats (n = 5) were treated with either OT (5 µg/kg), OTA (desGly-NH2d(CH2)5-[D-Tyr2, Thr4]-vasotocin 2 µg/kg), OT (5 µg/kg) plus OTA (2 µg/kg), or saline by sc injection, daily for 3 d. Animals were killed on the fourth day by CO2 inhalation; ventral prostates were immediately removed and divided before snap freezing in liquid nitrogen. One portion was used for isolation of total RNA by Trizol extraction, and second portion extracted for 5{alpha}-reductase activity assays.

Real-time RT-PCR
Expression of 5{alpha}-reductase I and 5{alpha}-reductase II in prostates from all treatment groups were investigated using quantitative real-time PCR. Eighty nanograms of total RNA was used to produce cDNA with Superscript RT system (Invitrogen Life Technologies) from random hexamers according to the manufacturer’s instructions. Two µl of reverse transcribed cDNAs were added to PCR mix containing 12.5 µl TaqMan universal mastermix (Applied Biosystems, Branchburg, NJ), 0.9 µmol·liter–1 of specific forward and reverse primers and 0.25 µmol·liter–1 of specific FAM reporter dye 5'-labeled probe (3'-TAMRA quenched) in a final reaction volume of 25 µl. Real-time PCR was then performed using the ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) with an initial denaturation step of 95 C for 10 min followed by 40 cycles of at 95 C for 15 sec (denaturation) and 60 C for 1 min (annealing and extension). Forward and reverse primers for the type I isoform were 5'-CTTGAGCCAGTTTGCGGTTT-3' and 5'-TTTTCTCAGATTCCTCAGGATGTG-3', corresponding to nucleotides 399–418 and 505–528 of rat 5{alpha}-reductase I mRNA, respectively, and sequence specific probe was 6FAM-TGAAGACTGG-TAMRA. 5{alpha}-Reductase II specific forward and reverse primers corresponding to nucleotides 213–233 and 313–341 of the rat 5{alpha}-reductase II mRNA, and probe were 5'-CAGGAGTTGCCTTCCTTTGTG-3', 5'-GTAAATAAATGTCCTGTGAAGTAATGTG-3', and 6FAM-CTTCGGACCGCCCGGGAATGT-TAMRA, respectively. Absolute standards (0.975–250 fg) prepared from purified cDNA identical to real-time PCR products were included on each plate to ensure equal efficiency of amplification between standards and PCR products generated in sample wells.

5{alpha}-Reductase activity assays
Prostate tissue was weighed and homogenized in 5 vol of 880 mmol·liter–1 sucrose, 1.5 mmol·liter–1 CaCl2, and protein concentration was determined. One hundred microliters of prostate homogenate were added to 300 µl of either 5{alpha}-reductase type 1 [0.01 mol·liter–1 phosphate buffer (pH 7.0)] or 5{alpha}-reductase type 2 [0.01 mol·liter–1 acetate buffer (pH 5.0)] buffer containing 0.5 mmol·liter–1 ß-nicotinamide adenine dinucleotide phosphate, 25 nmol·liter–1 [3H]-testosterone (specific activity 3.7 TBq per mmol·liter–1), 9.5 µmol·liter–1 testosterone, and incubated with agitation for 1 h at 37 C. Reactions were quenched with ice cold water, steroids extracted and separated by HPLC on a RP-18 octedecyl silica column (5 µm pore size, 4 mm x 250 mm; Merck KGaA, Darmstadt, Germany) with 40% (vol/vol) acetonitrile at a flow rate of 1 ml · min–1). Fractions were collected and the rate of conversion to DHT calculated from the peak area determined by liquid scintillation counting.

RIA of plasma and prostatic DHT
Steroids were extracted from plasma and prostate tissue by diethyl-ether and 70% methanol, respectively (9). DHT was measured in extracts according to the method of Jenkin and Nicholson (19). The limit of detection was 125 pg·ml–1 with an intraassay variation of 14.1%. All samples were measured in one assay.

Localization of 5{alpha}-reductase isoforms in the rat prostate
A high temperature antigen retrieval technique as described by Whittington et al. (16) was employed. Following retrieval, sections were washed twice with distilled water, once in Tris-buffered saline [25 mmol·liter–1 Tris, 150 mmol·liter–1 NaCl (pH 7.6)], Twice in Tris-buffered saline with 0.5% (vol/vol) Triton X-100 and finally once in Tris-buffered saline with 0.5% (vol/vol) Triton X-100 and 3% (wt/vol) BSA. Endogenous biotin was blocked in all sections by incubation with avidin (1 mg·ml–1) for 20 min, followed by incubation with biotin (0.1 liter–1) for 20 min. Sections were then rinsed in Tris-buffered saline before incubating overnight at 4 C with antisera raised against either 5{alpha}-reductase type I or 5{alpha}-reductase type II (12, 20) (diluted 1:75 in buffered saline with 0.5% (vol/vol) Triton X-100 and 3% (wt/vol) BSA). Sections were washed in Tris-buffered saline with 0.5% (vol/vol) Triton X-100 and 3% (wt/vol) BSA and then incubated with biotin-conjugated goat antirabbit IgG (Sigma, Poole, UK), diluted 1:200 in buffered saline with 0.5% (vol/vol) Triton X-100 and 3% (wt/vol) BSA for 1 h at room temperature. Incubation was followed by two washes in Tris-buffered saline and then incubation with a solution of avidin complexed with biotinylated horseradish peroxidase (ABC complex/HRP; Dako Ltd., High Wycombe, UK) for 30 min. Sections were washed three times in Tris-buffered saline followed by two washes in 25 mmol·liter–1 Tris (pH 7.6). Immunoreactive peptide was visualized with hydrogen peroxidase (2 mg·ml–1) and diaminobenzidine (0.7 ng·ml–1) in 0.06 mmol·liter–1 Tris-HCl. Sections were counterstained with hematoxylin before dehydration and mounting in distrene 80 dibutyl pthalate and xylene (DPX) medium (BDH Laboratory Supplies, Poole, Dorset, UK). Control sections were incubated with preimmune serum in place of the primary antibody.

Statistical analysis
Data are expressed as means ± SEM. Significant differences between treatment group means were tested for by one-way ANOVA. When significant differences were found, post hoc comparison of means by Tukey’s honestly significant difference method determined where differences occurred (21).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of OT receptor mRNA in the rat prostate
RT-PCR using primers specific for OT receptor generated amplicons of approximately 247 bp (Fig. 1Go) from RNA extracts of rat prostate. Sequence analysis of subcloned products from two individual rats and alignment to known sequences using the National Center for Biotechnology Information-Basic Local Alignment and Search Tool database confirmed the product to be a specific OT receptor cDNA with 100% homology to the rat sequence (15).



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  		FIG. 1. Identification of OT receptor (OTR) mRNA by RT-PCR in rat prostate. Similarly, cDNA from rat prostate was amplified using ß-actin specific primers to generate a predicted 741-bp amplicon as a positive RT-PCR control. Amplification of non-reverse-transcribed (no RT) prostate RNA using ß-actin and OTR primers served as negative controls.

 
Identification of OT receptor protein in the rat prostate
Western blot analysis identified an immunopositive band Mr 60 x 10–3, corresponding to that found in the rat uterus, in all samples of adult prostate (Fig. 2Go). A corresponding band was absent in extracts of liver.



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  		FIG. 2. Determination of the presence of OT receptor in rat prostate. A, Western blot analysis of rat prostate and uterus extracts for the presence of OT receptor. Blots were probed with a 1:2000 dilution of 020 antiserum. B and C, Binding isotherms, of rat prostate and testis extracts, respectively, for iodinated OTA (125I-OTA). Scatchard plots of bound/free (Bo/Lo) vs. bound ligand (Bo) are given as insets.

 
Immunocytochemical analysis of rat uterine sections as positive controls confirmed the specificity of the primary antibody with positive immunoreactivity being observed in the glandular tissue of the endometrium and the musculature of the myometrium (Fig. 3CGo). In the ventral prostate immunostaining was present in the glandular epithelial cells, often more intense at the lumenal border, and in the stromal tissue (Fig. 3DGo). This immunoreactivity was significantly reduced in sections incubated with antiserum preadsorbed with synthetic peptide (Fig. 3Go, E and F). No immunoreactivity was determined in sections of both uterus and prostate incubated with preimmune sera (Fig. 3Go, A and B).



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  		FIG. 3. OT receptor localization by immunocytochemistry and autoradiography. Five-micrometer-thin sections of rat: A, uterus incubated with preimmune serum; B, ventral prostate incubated with preimmune serum; C, uterus incubated with 1:100 dilution of 020; D, ventral prostate incubated with 1:100 dilution 020; E, uterus incubated with 1:100 dilution of 020 preadsorbed with immunogen; and F, ventral prostate incubated with 1:100 dilution of 020 preadsorbed with immunogen. Arrowheads indicate prostatic epithelium; arrows indicate stromal tissue. Scale bars, 10 µm.

 
Radioligand receptor binding assay
Analysis of 125I-OTA binding isotherms of homogenates of rat prostates revealed a saturable population of binding sites that adhered to a one-site binding model. The affinity of binding sites in the prostate determined as a Kd = 0.08 ± 0.011 nmol·liter–1 was similar to that determined for testis (Kd = 0.13 ± 0.02). Receptor density in the prostate was also similar to the testis with values of maximum binding capacity determined as 9.1 ± 0.52 fmol·mg–1 protein and 7.75 ± 0.49 fmol·mg–1 protein, respectively. No specific binding of 125I-OTA to homogenates of skeletal muscle was determined.

Effects of OT treatment on expression of 5{alpha}-reductase isoforms in the rat prostate
In the prostates of rats administered OT, significantly lower levels of 5{alpha}-reductase I mRNA (P < 0.01) were present compared with those of control, OTA or OT + OTA treatment groups (Fig. 4AGo). 5{alpha}-Reductase II mRNA was significantly increased (P < 0.01) in prostates of rats treated with OT compared with levels in prostates of rats in other treatment groups (Fig. 4BGo).



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  		FIG. 4. Real-time RT-PCR analysis of 5{alpha}-reductase isoforms I (A) and II (B) expression in the ventral prostates of adult male rats treated with OT, OTA, OT + OTA, or saline (Control). Data are expressed as mean (±SEM) amounts of cDNA per gram of total RNA template in reverse transcription reaction (**, P < 0.01; n = 5).

 
Effects of OT treatment on activity of 5{alpha}-reductase isoforms in the rat prostate
Activity of prostatic 5{alpha}-reductase I was significantly increased (P < 0.01) in rats treated with OT. The magnitude of this increase was significantly attenuated in the OT + OTA treatment group compared with OT alone (P < 0.05). However, OTA did not completely block the action of OT as activity was still significantly raised (P < 0.05) compared with both the control and OTA only treatment groups (Fig. 5AGo). 5{alpha}-Reductase II activity was significantly increased (P < 0.01) by OT but significantly decreased by OTA (P < 0.05) compared with control activity. Activity of 5{alpha}-reductase II in prostates of those rats treated with OT + OTA was not significantly different from controls (Fig. 5BGo).



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  		FIG. 5. Activity of 5{alpha}-reductase isoforms I (A) and II (B) in the ventral prostates of adult male rats treated with OT, OTA, OT + OTA, or saline (Control). Data are expressed as mean (±SEM), and differences between means are denoted by numbers where bars not sharing a number are significantly different (*, P < 0.05; **, P < 0.01; n = 5).

 
Effects of OT treatment on plasma and prostatic concentrations of DHT
Both plasma and prostatic concentrations of DHT were significantly greater (P < 0.05 and P < 0.01, respectively) in rats treated with OT than rats treated with vehicle alone. Furthermore, OT administration resulted in significantly lower concentrations of both plasma (P < 0.01) and prostatic DHT (Table 1Go).


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  		TABLE 1. Mean (±SEM) plasma and prostatic concentrations of DHT in rats

 
Effects of OT treatment on distribution of 5{alpha}-reductase isoforms in the rat prostate
Both type I and type II isoforms were localized to the cytoplasm of prostatic epithelium and stroma of the ventral prostate. In many cells, immunoreactivity was more intense around nuclei. Both the number of 5{alpha}-reductase type I immunoreactive cells, and intensity of signal appeared less in all sections from animals treated with OT compared with controls (Fig. 6Go, A and B). Staining was very dark in control sections both in the epithelium and stroma. In contrast, the number of 5{alpha}-reductase type II cells and intensity of signal were much greater in all sections of the OT-treated group in comparison to control animals (Fig. 6Go, C and D). There appeared to be no changes in distribution, although changes in immunoreactivity of both isoforms were more prominent in the epithelium. No immunoreactivity was determined in negative controls (not shown).



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  		FIG. 6. Immunolocalization of 5{alpha}-reductase isoforms in rat ventral prostate. Five-micrometer-thin sections incubated with 5{alpha}-reductase type I antiserum from A, control; B, OT treatment group and sections incubated with 5{alpha}-reductase type II antiserum from C, control; D, OT treatment group. Arrowheads indicate prostatic epithelium; arrows indicate stromal tissue. Scale bars, 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The generation of a cDNA by RT-PCR with sequence homology to OT receptor mRNA demonstrates that the OT receptor gene is expressed in the rat ventral prostate. The demonstration of an immunoreactive protein of similar in size to that previously reported for rat uterus (22) and mammary gland (23) by Western blot analysis, confirms that gene expression results in expression of protein. Radioligand binding assays demonstrate the presence of a single high affinity binding site. The affinity of this site is similar between prostate and testis and both measures are consistent with that reported by Bathgate and Sernia (24) (Ka 13.8 nmol·liter–1, equivalent to a Kd~0.07 nmol·liter–1) in the rat testis and previously published values for the uterus (25, 26).

The localization of the OT receptor by immunocytochemistry in the rat prostate to both stroma and glandular epithelium differs from that previously reported in primates where staining is predominantly in the stromal tissue (13, 14). This may reflect the different anatomical arrangements of the gland in these species or possibly that the peptide has differing functions in the rat and primate. However, recent work in our laboratory has localized OT receptor to the epithelium of the human prostate (27). In the rat prostate, OT has been demonstrated to promote epithelial growth by stimulating mitotic activity and reducing epithelial cell apoptosis (28), resulting in an increase in the epithelial cell number (29). The presence of receptors on epithelial cells would support this role.

The peptide also stimulates 5{alpha}-reductase activity in the rat prostate (10). Significantly, the present study demonstrates that the activity of both isoforms of 5{alpha}-reductase are increased by treatment with OT for 3 d, causing increased concentrations of prostatic DHT. This action was blocked (type II isoform) or significantly attenuated (type I isoform) by the presence of a specific OTA demonstrating that this action is specific. Interestingly, the findings also suggest that the mechanisms by which OT influence these enzymes differ. Although activity of 5{alpha}-reductase type I is increased, its gene expression is decreased. This is puzzling given that it has been demonstrated that increased DHT feeds forward to increase expression of both isoforms of 5{alpha}-reductase (11). It is possible that OT acts both at the protein and gene level of 5{alpha}-reductase type I.

It is known that the phosphorylation state of 5{alpha}-reductase plays an important part in the regulation of its activity in a number of tissues, including the prostate (30). Analysis of the peptide sequence for 5{alpha}-reductase type I predicts a tyrosine kinase phosphorylation site at residues 176–183. The OT receptor can be coupled to the G{alpha}i signal transduction protein (22), which activates tyrosine kinase (31) and hence OT could induce phosphorylation and activation of existing enzyme. Indeed, 5{alpha}-reductase activity in homogenates of testes and epididymides is increased in a dose-dependent manner by OT, suggesting that OT does activate existing enzyme (9). OT receptor can also be coupled to G{alpha}q/11 signal transduction protein (22), which activates protein kinase C (31). It is possible then that stimulation of MAPK by protein kinase C leads to down-regulation of 5{alpha}-reductase type I gene expression (32). These findings help to explain previous results that showed that when OT treatment is extended beyond 3 d 5{alpha}-reductase activity returns to control values (10). We suggest that this is because of down-regulation of the type I isoform gene expression, which subsequently leads to significantly lower levels of enzyme available for direct activation by OT-stimulated phosphorylation.

Increased activity of 5{alpha}-reductase type II in the prostates of OT-treated animals may also be because of activation by phosphorylation of existing enzyme. Analysis of its peptide sequence predicts a protein kinase C phosphorylation site at residues 250–253. Given that localization of 5{alpha}-reductase type II is predominantly stromal (12), and that OT receptors are also present in the stroma, then this mode of action is likely. The response of expression of type II isozyme is also consistent with feed forward regulation by DHT (11). We propose that the OT-induced increase in stromal 5{alpha}-reductase type II expression is modulated by the increased DHT produced in the prostatic epithelium by 5{alpha}-reductase type I. Increased 5{alpha}-reductase type II peptide is then activated by phosphorylation through the action of OT on its receptor in stromal tissue.

Changes in expression of both isoforms of 5{alpha}-reductase are supported by immunocytochemistry that demonstrated an increase in type II but a decrease in type I following treatment with OT. This immunolocalization, however, is slightly different to that previously described in the rat (12). In that study, localization of both isoforms was described in regenerating ventral prostate stimulated by testosterone supplementation following castration and as such is not directly comparable. Indeed, in the human it has been demonstrated that both type I and type II isoforms are expressed in stromal and epithelial tissues (33).

In conclusion, a single specific and active OT receptor is expressed in the rat ventral prostate and is present in both the glandular epithelium and the stroma. This distribution is consistent with regulation of both 5{alpha}-reductase isoforms by OT in the rat prostate. OT increases activity of both 5{alpha}-reductase types I and II. However, regulation of isoforms at the gene level is different with type I being down-regulated, whereas type II is up-regulated after treatment with OT.


   Acknowledgments
 
We thank Maurice Manning (Medical College of Ohio, Toledo, OH) for the kind gift of OTA, David Russell (University of Texas, Dallas, TX) for the kind gift of 5{alpha}-reductase antisera, and Maree Gould (University of Otago, Dunedin, New Zealand) for technical assistance.


   Footnotes
 
This work was funded in part by an Otago School of Medical Sciences Deans Award.

Abbreviations: DHT, Dihydrotestosterone; Kd, dissociation constant; OT, oxytocin; OTA, OT antagonist.

Received June 3, 2004.

Accepted for publication September 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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