Mesotocin Receptor Gene and Protein Expression in the Prostate Gland, but Not Testis, of the Tammar Wallaby, Macropus eugenii¹

^a Department of Zoology, University of Melbourne, Parkville, Victoria 3052, Australia ^b Division of Reproductive Sciences, IHF Institute for Hormone&Fertility Research, University of Hamburg, 22529 Hamburg, Germany

	ABSTRACT

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Evidence suggests that systemic oxytocin (OT) causes contractions of the prostate gland during ejaculation in eutherians, although functional OT receptors in this tissue have not been identified. Male marsupials secrete mesotocin (MT) from the pituitary and have relatively large, muscular prostate glands, so we examined MT receptors (MTRs) in the reproductive tract of the male tammar wallaby at the mRNA and protein level. We first obtained a partial (588 base pair) sequence of the tammar MTR cDNA that showed high homology to eutherian OT receptors (74–77%) and low homology to vasopressin receptors (38–52%). Analysis by reverse transcription-polymerase chain reaction demonstrated MTR mRNA in the adult, juvenile, and pouch young prostate and epididymis, but not testis. MTR transcripts were observed in the smooth muscle layers surrounding the urethral lumen and in the fibromuscular capsule. There was a single high-affinity ¹²⁵I-D(CH₂)₅[Tyr(Me)², Tyr⁴, Orn⁸, Tyr-NH₂⁹]-vasotocin (¹²⁵I-OTA) binding site in the adult prostate. Competitive binding assays revealed identical ligand-binding profiles to the myometrium MTR (OTA > OT = MT > arginine vasopressin [AVP] antagonist > AVP). A lower-affinity ¹²⁵I-OTA-binding site was present in the testis, with ligand-binding profiles indicating binding to vasopressin receptors. MTR concentrations in the prostate were 8-fold lower than concentrations in the myometrium. Our data demonstrate the presence of an MTR gene and functional receptor protein in the prostate gland, but not the testis, of the tammar. Localization of MTRs to the smooth muscle fibers in the capsule and surrounding the urethral lumen suggests a contractile function for MT during ejaculation.

	INTRODUCTION

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It is now well established in eutherians that the peptide hormone oxytocin (OT) is synthesized locally in the testis and may influence steroidogenesis and growth of the prostate gland by increasing 5-reductase activity [1, 2]. Treatment with OT in castrated rats enhances the size of the stromal prostatic glands and the volume of the epithelial cells lining these glands by stimulating mitotic activity and causing a decrease in apoptosis [3, 4]. These functions probably depend on a paracrine hormone-receptor interaction in the male reproductive tract. However, evidence also suggests that systemic OT controls muscular contractility in the prostate gland and epididymis, thereby facilitating ejaculation [2]. One mechanism of action may be a direct effect on the smooth musculature; OT stimulates prostatic contractions in a dose-dependent manner in vitro and increases muscular tone in the rat, dog, and guinea pig [5, 6]. There are very few data on functional OT receptors in eutherian male accessory glands. Agmo [7] reported uptake of iodinated OT by the prostate and seminal vesicles in the rat. Recently, an OT receptor gene transcript was demonstrated in the marmoset prostate [8]. OT receptor immunoreactivity was also observed in the basal part of the glandular epithelium, with sporadic staining in the stromal tissue [8], but not associated with smooth muscle cells.

Unlike eutherians, most marsupials secrete mesotocin (MT), which differs from OT by the substitution of isoleucine for leucine at position 8 [9]. Immunoreactive MT is found in Leydig cells of the possum Trichosurus vulpecula and bandicoot Isoodon macrourus testis, with concentrations similar to those found in the human and rat [9]. MT and OT have both been localized in the prostate gland of the bandicoot [10]. However, target tissues for these neuropeptides have not been demonstrated. The prostate and paired Cowper's glands are the only accessory glands in male marsupials. The disseminate prostate is generally divided into distinct segments: anterior, central, and posterior. Each segment consists of glandular tissue surrounded by the smooth muscle layer of the urethra [11]. A particular feature of the prostate gland in the adult tammar wallaby, Macropus eugenii, is its size and weight, which reaches 120 g annually during the breeding season (December–March) and represents 1.3% of the total body weight [12]. A similar prostatic hypertrophy occurs in the possum during its breeding season. The size and appearance of the prostatic glands in each segment vary between breeding and nonbreeding animals. Glands are larger in diameter and the height of the epithelium lining is greater in breeding animals [12]. As the prostate also has a thick smooth muscle layer in the outermost capsule [11], it is possible that MT may be important in male tammars to stimulate contractions of the prostate as well as influence growth during the breeding season, thereby facilitating ejaculation. We examined the presence of MT receptor (MTRs) at both the mRNA and protein level in the reproductive tract of the male tammar wallaby to establish whether or not MT may act on the prostate.

	MATERIALS AND METHODS

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Testis, prostate gland, and epididymis tissues were obtained from 5 adult males in the breeding season (January–February) and 3 juvenile male tammar wallabies (8–12 mo old) shot in the wild on Kangaroo Island (South Australia) under permit number A23892-01 from the South Australia National Parks&Wildlife Services. The prostate glands from 2 pouch young (140 and 148 days old) were kindly provided by Professor Marilyn Renfree (University of Melbourne). Tissues were rapidly frozen in liquid nitrogen, transported to Melbourne on dry ice, and stored at -80°C. Pieces of adult testis and prostate were also fixed in 4% paraformaldehyde overnight at 4°C, washed twice in single-strength PBS, and stored in 70% ethanol until embedded in paraffin wax.

MTR Gene Expression in Male Reproductive Tract Tissues

A partial sequence of the tammar MTR gene was obtained using RNA extracted from adult prostate and reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from 150–200 mg tissue according to the Chomczynski and Sacchi method [13]. Quantity and quality of the RNA were assessed by spectrophotometry at 260 and 280 nm and ethidium bromide staining after agarose gel electrophoresis. First-strand cDNA was synthesized using 5 µg total RNA, 500 ng/µl oligo(dT)_12–18 primer (Pharmacia Biotech, Freiberg, Germany), and 200 U Superscript II reverse transcriptase enzyme (Gibco BRL, Eggenstein, Germany) in a total volume of 20 µl [14]. Two microliters of this cDNA template was then used in a touchdown PCR reaction (50 µl volume) with 0.2 U Taq DNA polymerase (Promega, Heidelberg, Germany) and 100 ng/µl oligonucleotide primers (forward primer 5'-ACCTGGTGGCAGTGTTTCAGGT and reverse primer 5'-AAGAAGAAAGGCGTCCAGCACA-CGA) that correspond to highly conserved regions in exon 2 of the human and bovine OT receptor sequences [15, 16]. The PCR reaction proceeded for 40 cycles of 1-min denaturation at 95°C, 1 min at the annealing temperature (annealing temperatures decreased from 65°C to 55°C), and 1-min extension temperature at 72°C in a Biometra (Gottingen, Germany) Trio-Thermo block. As a negative control, water replaced the cDNA template. The resulting PCR products were subjected to agarose gel electrophoresis followed by ethidium bromide staining. A single PCR product of 588 base pairs (bp) was obtained, eluted from the agarose gel using QIAEXII gel extraction kit (Qiagen GmbH, Hilden, Germany), and subcloned into a pGEM-T Vector (Promega). Subsequent nucleic acid sequence analysis (T7 sequencing kit; Pharmacia Biotech) confirmed that this subclone consisted of 196 amino acids corresponding to a putative OT-like receptor. Homologous tammar-specific primers and various touchdown PCR programs were then designed to obtain multiple (n = 7) independent subclones from different tissues, including endometrium from a late-pregnant female tammar.

MTR gene expression was then analyzed in adult, juvenile, and pouch young tissues using RT-PCR with tammar-specific MTR oligonucleotide primers (forward 5'-TCGTCAAG TATCTGCAGGTGG, reverse 5'-CCTCACAGGTAACAGATGAGG) yielding a product of 400 bp. As these primers do not span an intron, total RNA (20 mg) was subjected to DNase I treatment (2 U RQ1; Promega, Annandale, NSW, Australia) prior to first-strand cDNA synthesis to degrade genomic DNA contaminants. In a further control experiment, the single-stranded cDNA was treated with RNase (1 U RNase A, 20 U RNase T1; Ambion, Bresatec, Adelaide, SA, Australia) and then subjected to PCR under the same conditions as untreated cDNA. The quality of RNA and cDNA synthesis was demonstrated in parallel RT-PCR reactions that assessed expression of glyceraldehyde phosphate dehydrogenase (GAPDH). Subsequent Southern blotting used an internal 40-mer oligonucleotide probe, end-labeled with [-³²P]ATP (Bresatec) and T4 polynucleotide kinase (New England BioLabs, Genesearch Pty Ltd., Arundel, Queensland, Australia) to a specific activity of 3.7 x 10⁷/mg DNA. The PCR products were transferred onto nylon membranes (Hybond-N; Amersham Life Science, Castle Hill, NSW, Australia) and hybridized overnight at 65°C with radiolabeled probe (2.8 x 10⁶ cpm) as described previously [14]. Membranes were washed twice in double-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate)/0.1% SDS at room temperature for 5 and 15 min and once in 0.2-strength SSC/0.1% SDS at 65°C for 15 min; they were then exposed to film (Kodak BioMax, Amersham) with a single intensifying screen at -70°C for 10 h.

In Situ Hybridization

Digoxigenin-labeled riboprobes were prepared using a DIG RNA labeling kit (Boehringer Mannheim, Nunawading, Victoria, Australia) according to the manufacturer's instructions. Antisense and sense cRNA probes were generated from a linearized plasmid pGEM-T vector containing an NCOI-NOTI 300-bp PCR fragment of tammar MTR DNA. Paraffin-embedded tissue sections (7 mm) were mounted on Superfrost Plus slides (Menzel; HD Scientific, Bayswater, Victoria, Australia), hydrated in xylene and 100%, 90%, 70%, and 50% ethanol, and treated with 0.2 N HCl for 8 min to inactivate alkaline phosphatase. The sections were incubated in 10 mg/ml proteinase K (Boehringer Mannheim) for 20 min at 37°C and postfixed in 3% paraformaldehyde, 0.1 M PBS (pH 7.4) for 15 min before acetylation in 0.2% acetic acid anhydride in 0.1 M triethanolamine, pH 8.0. Tissue sections were prehybridized for 2 h at 45°C in 50 ml hybridization solution containing 50% deionized formamide, 20 mM Tris-HCl (pH 7.5), single-strength Denhardt's solution, 300 mM NaCl, 100 mM dithiothreitol, 1 mM EDTA, 100 mg/ml Poly A (Boehringer Mannheim), 500 mg/ml Escherichia coli tRNA, and 500 mg/ml salmon sperm DNA. The prehybridization solution was then replaced with 50 ml fresh hybridization solution containing 10% dextran sulfate and 1 mg/ml digoxigenin-labeled antisense riboprobe. The tissue sections were covered with agarose gel bond support medium (Gel Bond Biozyme, 0.2 mm; Edwards Instrument Commpay, Narellan, NSW, Australia) and incubated at 45°C for 16 h in a moist chamber. After hybridization, the sections were washed in double-strength SSC for 15 min; they were then washed in 300 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM dithiothreitol, and 50% formamide at 37°C for 10 min and treated with RNase A (20 mg/ml) at 37°C for 30 min. They were next washed in double-strength SSC and 0.1-strength SSC at room temperature for 10 min each time and then in 0.1-strength SSC at 45°C for 15 min. The hybridized probe was detected using a Nucleic Acid Detection kit (Boehringer Mannheim). Slides were rinsed in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA, counterstained with 0.2% methyl green, and examined under brightfield microscopy. Controls consisted of 1) hybridization with the sense riboprobe and 2) RNase A pretreatment (20 mg/ml) at 37°C for 30 min before hybridization.

Characterization of the MTR in Male Reproductive Tract Tissues

Adult male prostate and testis tissues were weighed before homogenization in 20 (v:w) 10 mM Tris-HCl containing 1.5 mM EDTA (pH 7.4). The homogenates were centrifuged at 1000 x g for 15 min; supernatants were collected and recentrifuged at 120 000 x g for 30 min. The resulting microsomal fraction pellets were resuspended in 50 mM Tris-HCl, 5 mM MgCl₂ (pH 7.6) and then diluted based on the original tissue weights to a final dilution of 1:10. Diluted tissue suspensions were used immediately in the radioreceptor assay. Protein concentrations of the resuspended pellets were measured by the dye-binding method of Bradford [17] using BSA as the protein standard. Assay protein concentrations were in the range of 5-100 mg/tube, which was within the range where specific binding is linearly correlated with protein concentration. Radioreceptor assays were carried out as described by Parry et al. [18] using ¹²⁵I-D(CH₂)₅[Tyr(Me)², Tyr⁴, Orn⁸, Tyr-NH₂⁹]-vasotocin (¹²⁵I-OTA) as the labeled ligand. The receptor assay mixture consisted of 0.1 ml diluted tissue suspension, 0.1 ml ¹²⁵I-OTA (150 000 cpm/ml), and 0.1 ml assay buffer (50 mM Tris-HCl, 5 mM MgCl₂, 0.2% BSA, pH 7.6) containing a range of 0.005-1 pmol/tube unlabeled OTA (kindly provided by Dr. Maurice Manning, Medical College of Ohio, Toledo, OH). Nonspecific binding was determined by the addition of 40 pmol/tube OTA. The data were examined by nonlinear regression analysis using the Ligand computer program (Bethesda, MD) to obtain the binding affinity (K_a) and the receptor content (R_o) for radiolabeled ligand binding. Competitive binding radioreceptor assays determined ligand specificity using MT, OT, arginine vasopressin (AVP), lysine vasopressin (LVP) (all from Bachem UK Ltd., Saffron Walden, Essex, UK), and a vasopressin V1 receptor antagonist (D(CH₂)₅, Tyr (Me)²-AVP; MC); kindly provided by Dr. Maurice Manning, Medical College of Ohio, Toledo, OH). The interaction of each peptide with ¹²⁵I-OTA was expressed as relative displacement curves B/B_o versus log concentration and fitted with sigmoid curves.

	RESULTS

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MTR Gene Expression in Male Reproductive Tract Tissues

Sequence analysis of the various PCR fragments obtained using tammar MTR-specific oligonucleotide primers and various tissues produced a consensus nucleotide sequence of 588 bp, which encoded a peptide of 196 amino acids (Fig. 1). This derived amino acid sequence showed a high homology (74–77%) to the region extending from the II to the VI putative transmembrane domain in all eutherian OT receptor genes published but low homology to vasopressin receptors (38–52%). Of particular interest is the identification of highly conserved regions in the III transmembrane domain and the first and second extracellular loops (Fig. 1).

View larger version (66K): [in this window] [in a new window]   FIG. 1. The partial 196-amino acid sequence of the putative tammar MTR cDNA. Areas of homology compared with the rat, bovine, human, and sheep OT receptor gene sequences are indicated with asterisks. The positions of the putative transmembrane domains are indicated above the arrows with Roman numerals. Of particular interest is the identification of highly conserved regions in the III transmembrane domain and in the first and second extracellular loops.

RT-PCR analysis revealed a single PCR product of 400 bp expressed in the prostate gland of the adult, juvenile, and pouch young and also in the epididymis of the adult and juvenile tammar (Fig. 2a). A strong positive signal was present in the myometrium of the late-pregnant female tammar. No MTR transcripts were observed in the testis. All samples showed strong positive signals for GAPDH mRNA (Fig. 2a), demonstrating equivalent quality of reverse transcription reactions. No MTR transcripts were detected if water replaced the cDNA template in the PCR reaction. The lack of a positive signal in those prostate samples pretreated with RNase prior to the PCR reaction (data not shown) indicated that any PCR products arose from cDNA. Specificity was confirmed by sequence analysis and hybridization with a gene-specific internal oligonucleotide probe. Strong positive signals were observed in the myometrium, prostate, and epididymis of the adult tammar compared with weaker signals in equivalent tissues in the juvenile and pouch young males (Fig. 2b). A weak positive signal was also present in the testis.

View larger version (75K): [in this window] [in a new window]   FIG. 2. a) MTR gene transcripts were detected in the prostate (P) and epididymis (E) of the adult (lanes 2 and 3), juvenile (lanes 5 and 6), and pouch young (PY: lane 7) and also in the myometrium (myo) of the late-pregnant female (lane 8). However, no MTR transcripts were detected in the testis (T) of either the adult or juvenile (lanes 1 and 4) or in the PCR reaction in which water replaced the cDNA template (*). All samples showed strong positive signals for GAPDH mRNA, demonstrating equivalent quality of reverse transcription reactions. MW: 100-bp DNA marker. b) Southern hybridization analysis of RT-PCR products specific for MTR mRNA. The membrane was hybridized with a radiolabeled tammar MTR oligonucleotide probe and exposed to film for 10 h. Control: Lane 8 (*), where water replaced the cDNA in the PCR reaction.

Localization of MTR Transcripts in the Prostate

The tammar prostate surrounds the urethra as it leaves the bladder and consists of numerous tubular glands arranged concentrically around the urethra (Fig. 3a). A dense layer of smooth muscle separates the prostatic glands from the urethral lumen (Fig. 3b). Smooth muscle cells are also present in the stromal tissue between the prostatic glands (Fig. 3c). These glands are lined with columnar epithelial cells that vary in height between prostate segments; the lumina contain secretory material with small globular bodies (Fig. 3c). The outermost layer of the prostate is a fibroelastic capsule with smooth muscle fibers on the inner aspect. Blood vessels are present in this outer connective tissue layer (Fig. 3d). MTR transcripts were identified in the smooth muscle layer surrounding the urethral lumen (Fig. 4a) and also in the outer fibroelastic capsule. There was no evidence of MTR mRNA in the stromal tissue between the glands, but there was some positive staining in the basal part of the glandular epithelium (Fig. 4b). Sections that were hybridized with the sense probe, or pretreated with RNase before hybridization, showed no positive signals, and there were no specific MTR transcripts in the testis (Fig. 4c).

View larger version (148K): [in this window] [in a new window]   FIG. 3. Histology of the adult tammar prostate (hematoxylin- and eosin-stained sections). a, b) Numerous tubular glands (G) are arranged concentrically around the urethral lumen (UL), with a dense layer of smooth muscle (SM) between the prostatic glands and urethral lumen. c) Smooth muscle cells (arrowheads) are present in the stromal tissue between the prostatic glands, which are lined with columnar epithelial cells (EC). d) The outermost layer of the prostate is a fibroelastic capsule (FC) containing smooth muscle fibers and blood vessels (V). a) x7.5; b) x50; c) x100; d) x62.5 (reproduced at 70%).

View larger version (138K): [in this window] [in a new window]   FIG. 4. Localization of MTR transcripts using in situ hybridization with a digoxigenin-labeled MTR antisense probe. a, b) MTR transcripts (arrowheads) are present in the smooth muscle layer surrounding the lumen and also in the basal part of the glandular epithelium. c) No positive signals are evident in the adult testis. a, b) x50; c) x25 (reproduced at 68%).

Characterization of the MTR in Male Reproductive Tract Tissues

Scatchard plots of ¹²⁵I-OTA binding to matched samples of adult tammar prostate and testis were linear, indicating a single class of binding site that was confirmed by analysis with the Ligand program. The mean binding affinities (K_a) were similar in the prostate (2.1 ± 0.18 L/nmol: mean ± SEM, n = 8) and testis (1.3 ± 0.4 L/nmol). The specificity of ¹²⁵I-OTA binding was assessed by competitive displacement studies with various OT and vasopressin receptor agonists and antagonists (Fig. 5). In the prostate gland and myometrium, MT and OT showed relatively high and equal binding affinities for the ¹²⁵I-OTA-binding site, whereas AVP, LVP, and MC had much lower binding affinities. In general, the ligand-binding affinity was in the order of decreasing affinity OTA > MT = OT > MC > AVP = LVP, which is indicative of binding to an OT-like receptor. In the testis, however, LVP, AVP, and the vasopressin receptor antagonist MC had much higher binding affinities for the ¹²⁵I-OTA-binding site compared with MT and OT (Fig. 5c), suggesting binding to vasopressin-like receptors but not MTR. Concentrations of MTR in the adult prostate gland (27.7 ± 3.0 fmol/mg protein: n = 4) were 8-fold lower than those in the myometrium of the Day 22 pregnant female tammar but were higher than receptor concentrations in the median vagina and endometrium at the same stage of pregnancy. MTR concentrations were marginally higher, although not statistically different (ANOVA, p = 0.08), in the central portion of the prostate as compared with the anterior region.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Competitive displacement of 125I-OTA binding in the tammar gravid myometrium (a), prostate (b), and testis (c) by MT, OT, AVP, LVP, and MC.

	DISCUSSION

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We obtained a partial (588 bp) nucleotide sequence of a putative MTR cDNA in an Australian marsupial, the tammar wallaby, that showed high homology to eutherian OT receptors and low homology to vasopressin receptors. The 196-amino acid MTR fragment corresponds to the region extending from the II to the VI putative transmembrane domain in all eutherian OT receptor genes published. Our data highlight regions of highly conserved amino acids in the OT receptor gene that are important for its biological function. All the amino acids that have been shown from mutagenesis studies to be important for OT receptor binding and activation are conserved in the tammar MTR [19]. In particular, Phe 10 in the MTR sequence (which corresponds to Tyr 115 in the rat V1a receptor), Asp in AVP V2 receptors, and Phe in all OT receptors are essential for the selectivity of OT and vasopressin receptors for their respective ligands [19]. This is further evidence that our partial MTR sequence represents an authentic OT receptor. In contrast, there was relatively large variation in the third intracellular loop, which is thought to be the site of G-protein coupling. As suggested by Ivell et al. [20], variations in some regions of the OT receptor have been tolerated through its evolution and do not appear to affect OT binding or receptor activation.

Analysis of male reproductive tract tissues by RT-PCR demonstrated MTR transcripts in the prostate gland and epididymis, but not testis, of both adult and juvenile males. Transcripts were also detected in the prostate of pouch young aged between 4 and 5 mo. These findings are not in agreement with recent work in the marmoset monkey showing that OT receptor mRNA was strongly expressed in the adult testis but only weakly expressed in the epididymis, vas deferens, and prostate [8]. Our data at the mRNA level were confirmed at the protein level by radioreceptor assays that showed a high-affinity MTR in the prostate, but not the testis, of the male tammar wallaby. We previously characterized an MTR with ligand-binding specificities similar to those of the eutherian OT receptor in the tammar myometrium [18]. In the current study, we showed that the affinity (K_a) of the tammar prostate receptor for ¹²⁵I-OTA was similar to that of the uterine MTR. The prostate MTR was not highly selective for MT compared with OT and showed a low affinity for an AVP receptor agonist and antagonist. The relatively high binding affinities of both MT and OT for the ¹²⁵I-OTA-binding site, and the low affinities of AVP and the vasopressin receptor agonist and antagonist, confirmed that the ¹²⁵I-OTA-binding site in the tammar prostate represents an authentic MTR. Concentrations of MTRs in the anterior and central portion of the prostate were considerably lower than those of MTRs in the myometrium but were comparable with receptor concentrations in other female tissues such as the median vagina and endometrium. As MTR transcripts were found only in the smooth muscle layers surrounding the urethral lumen and in the outer fibroelastic capsule, low MTR protein concentrations may be due to a dilution effect from the glandular tissue that makes up the bulk of the prostate.

The lack of functional MTRs in the tammar testis suggests that MT does not influence testicular function in marsupials. Although we showed a high-affinity binding site for ¹²⁵I-OTA in the tammar testis, our data suggest that it is binding to an AVP-like receptor, because we demonstrated that the endogenous ligand LVP, as well as AVP, and a vasopressin receptor antagonist all had much higher binding affinities for the ¹²⁵I-OTA-binding site compared with MT and OT. However, without the corresponding mRNA data for tammar vasopressin receptors, we cannot be certain that such receptors are found in the testis of this marsupial. Bathgate and Sernia [21] were unable to detect MTRs in the possum testis but showed specific AVP receptors similar in density to AVP V1a receptors in the rat testis. Interestingly, the possum testis vasopressin receptor also showed a relatively high affinity for ¹²⁵I-OTA (0.22 L/nmol), and furthermore, in competitive displacement studies, vasopressin agonists and antagonists were able to displace this binding with much higher affinity than OT or MT. The AVP-binding sites in the possum showed marked differences in ligand-binding characteristics from rat AVP V1a and V2 receptors, and this may account for the variation in ¹²⁵I-OTA-binding affinities between marsupials and eutherians for the AVP receptor.

Although immunoreactive MT and OT have been reported in Leydig cells, within seminiferous tubules and in the dorsal portion of the prostate gland in some male marsupials [9, 10], neither the tammar testis nor prostate gland synthesizes MT [22]. MT has been extracted in microgram quantities from the pituitary of male tammars and possums, and its release in the latter species is stimulated by hypertonic stress and hemorrhage [23]. It is therefore likely that MT released into the peripheral circulation is the main source of peptide acting upon prostate MTRs. The blood supply to the possum prostate arises from a network of arteries and veins surrounding the neck of the bladder, with a large number of small blood vessels radiating into the body of the prostate [24]. A similar arrangement in tammars would enable peripherally circulating MT access to the prostatic parenchyma. Plasma MT concentrations in conscious male tammars do not differ from basal concentrations in pregnant females, but these data were obtained from daily blood samples [25]. It remains to be demonstrated whether or not MT release is associated with male sexual behavior coincident with ejaculation.

Male tammars reach sexual maturity between 18 and 20 mo of age when there is rapid growth of the testes. Thereafter, there is no seasonal increase in weight or change in morphology in either the testis or epididymis, and spermatogenesis occurs all year round. The tammar prostate develops between Days 25 and 60 postpartum and is largely under the influence of androgens and dihydrotestosterone [26, 27]. But there is a clear increase in weight of the adult prostate during the breeding season that is attributed to the seasonal fluctuation in plasma testosterone concentrations [12]. It is interesting to note that the MTR hybridization signal was stronger in the adult prostate compared with the juvenile and pouch young. This suggests the presence of higher numbers of MTRs in the adult prostate compared with that of prepubertal animals and may limit the role of MT in the male reproductive tract to adults. The distribution of MTR transcripts in the smooth muscle layers of the tammar prostate suggests that MT may affect ejaculation. The tammar male generally mates with one female; copulation takes on average 8 min, during which time the male will ejaculate between 10 and 50 ml semen into the female. Anesthetized electroejaculated males produce up to 20 ml semen [28]. Semen coagulates shortly after ejaculation; and 40 min after copulation, spermatozoa are concentrated within the two cervical canals. Given the relative size of the prostate (120 g) and the volume of ejaculate, coordinated contractions of the prostate smooth musculature may be essential to ensure successful mating and aid movement of spermatozoa into the female reproductive tract.

Our data confirm that the prostate may represent a target tissue in a putative MT reflex arc in male marsupials. We demonstrated MTR transcripts and protein in the prostate gland, but not the testis, of the tammar. Localization of MTRs to the smooth muscle fibers in the capsule and surrounding the urethral lumen suggests that MT may be important in male tammars to stimulate contractions of the prostate, thereby facilitating ejaculation.

	ACKNOWLEDGMENTS

 
The authors are especially grateful to Richard Ivell (IHF Institute for Hormone&Fertility Research, Hamburg, Germany) for providing laboratory facilities and funding to complete the sequencing work. We thank the Alexander von Humboldt Foundation for their support. We thank Marilyn Renfree for the pouch young tissues and for providing laboratory facilities in the Department of Zoology, University of Melbourne. We also thank David Paul for photography and Chris Butler for advice and criticism with the manuscript.

	FOOTNOTES

 
¹ Research was supported by a Clive&Vera Ramaciotti Research Grant, a University of Melbourne Collaborative Project Grant, and Grant Iv7/1-3 from the Deutsche Forschungsgemeinschaft. L.J.P. is currently an Australian Research Council Postdoctoral Fellow. Both L.J.P. and R.A.D.B. were recipients of Alexander von Humboldt Research Fellowships in Hamburg.

² Correspondence. FAX: 61 3 9344 7909; l.parry{at}zoology.unimelb.edu.au

Accepted: June 25, 1998.

Received: March 25, 1998.

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