Estrogens, But Not Androgens, Regulate Expression and Functional ...

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Endocrinology 143(11):4271– 4280 Copyright © 2002 by The Endocrine Society doi: 10.1210/en.2002-220384

Estrogens, But Not Androgens, Regulate Expression and Functional Activity of Oxytocin Receptor in Rabbit Epididymis SANDRA FILIPPI, MICHAELA LUCONI, SIMONE GRANCHI, LINDA VIGNOZZI, SAVERIO BETTUZZI, PAOLA TOZZI, FABRIZIO LEDDA, GIANNI FORTI, AND MARIO MAGGI Andrology Unit (S.F., M.L., S.G., L.V., G.F., M.M.), Department of Clinical Physiopathology, Department of Pharmacology (F.L.), University of Florence, Florence 50139, Italy; Azienda Ospedaliera Careggi (P.T.), Florence 50139, Italy; and Section of Biological Chemistry (S.B.), Department of Biomedical Science, University of Modena and Reggio Emilia, Modena 41100, Italy Previous binding and contractility studies indicate that oxytocin (OT) receptors are present in rabbit epididymis. To investigate the effect of changing endocrine milieu on OT responsiveness, we induced hypogonadism (hypo) in rabbits with a single administration of a long-acting GnRH analog, triptorelin, and we replaced hypogonadal rabbits with different sex steroids. After 2 months from triptorelin administration, testosterone (T) plasma levels were decreased and OT responsiveness abolished. Administration of T to hypo rabbits restored T plasma levels but not OT sensitivity. Because Western blot analysis indicated that both estrogen receptors and aromatase are expressed in the epididymis, we treated hypo rabbits with estradiol valerate (E2v). E2v not only completely

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XYTOCIN (OT) IS A HORMONE equally present in the posterior pituitary of both sexes (1, 2). However, its role has been defined only in the female: it regulates uterine activity and the milk ejection reflex (3). Therefore, OT has a pivotal importance in allowing reproduction and offspring care. Conversely, the physiological function of OT in the male is rather unclear. Several evidences indicate that OT might be involved in another relevant aspect of reproduction: the ejaculatory process. In fact, OT concentration increases in peripheral circulation during sexual arousal and orgasm (4 – 8), and pharmacological administration of OT increases the number of ejaculated sperm in several animal species (9 –16), including humans (17). This action of OT seems to be mediated by the activation of an OT receptor (OTR) present in the epididymis (17–20) that stimulates in vivo (21, 22) and in vitro (17, 23) its motility. The epididymis is the portion of the genital tract more proximal to the male gonad that plays an important role in the storage and maturation of spermatozoa produced by the testis. Sperm release from the epididymis is essentially promoted by the contractile activity of its smooth muscle cells. These contractile cells in the caput form a loose layer around tubules but in the cauda are organized in three distinct layers. We recently observed that in human epididymis OTR im-

Abbreviations: CC, Corpus cavernosum; E2, estradiol; E2v, estradiol valerate; ER, estrogen receptor; ET-1, endothelin-1; ␥-ACT, ␥-nonmuscle actin; NA, noradrenaline; OT, oxytocin; OTR, oxytocin receptor; Phe, phenylephrine; T, testosterone; TAM, tamoxifen; TTBS, Tween Tris buffer saline.

restored OT responsiveness but also even amplified it. Accordingly, Northern and Western blot analysis indicated that both OT receptor gene and protein were strongly induced by E2v but not by T. Surprisingly, also the class I estrogen receptor antagonist, tamoxifen restored OT sensitivity in hypo rabbits. To verify whether endogenous estradiol is involved in the regulation of OT receptor responsiveness, we treated intact rabbits with an aromatase inhibitor, letrozole. Blocking aromatase activity almost completely abolished OT sensitivity. These findings suggest a new function of estrogens in the male: regulation of OT responsiveness in epididymis. (Endocrinology 143: 4271– 4280, 2002)

munoreactivity was indeed present in the smooth muscle cells of the entire epididymis but was also localized in epithelial cells of the caput (17). In primary culture of epithelial cells from rabbit epididymis, we found (17) that OT promoted a dose-dependent release of another potent stimulator of epididymal motility, endothelin-1 (ET-1) (24). Hence, the contractile activity of OT in male epididymis in part is due to its direct effect on smooth muscle cells and in part mediated by the release of ET-1. In fact, blocking the ETA subtype of ET-1 receptor with a specific antagonist (BQ123) significantly reduced OT responsiveness in rabbit epididymis (17). Because there is strong evidence that sex steroids (androgens ⫹ estrogens) produced by the testis have an essential role in the differentiation and functional activity of the epididymis (25–28), in the present study, we investigated the effect of changing endocrine milieu on the expression and functional activity of OTR in the epididymis. Therefore, this study reports for the first time the demonstration of a new function of estrogens in the male: regulation of OTR in epididymis. Materials and Methods Chemicals Noradrenaline (NA), phenylephrine HCl, oxytocin, tamoxifen (TAM), [Thr4, Gly7] oxytocin, [deamino-Cys1, d-Arg8]-vasopressin (DDAVP), reagents for SDS-PAGE and peroxidase-conjugated antimouse, and rat secondary antibodies were purchased from Sigma (St. Louis, MO). (d(CH2)51, Tyr(Me)2, Orn8)-oxytocin, and (Phe2, Orn8)oxytocin were purchased from Bachem AG (Bubendorf, Switzerland). ET-1 was purchased from Calbiochem (La Jolla, CA). Testosterone en-

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anthate and estradiol valerate were supplied by Schering AG (Berlin, Germany); BM enhanced-chemiluminescence system was purchased by Roche Diagnostics (Milan, Italy). Reagents for protein measurement were from Bio-Rad Laboratories, Inc. (Hercules, CA). The IgG mouse monoclonal antibody 3–12 raised against the SVWDANAPKEAS sequence of human OTR (298 –309) was a kind gift of Dr. T. Kimura (Department of Obstetrics and Gynecology, Osaka, Japan); the monoclonal rat H222 antibody against the estrogen receptor was a kind gift of Prof. G. Greene (University of Chicago, Chicago, IL); rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase was a generous gift of Dr. C. Yarborough (Hauptman-Woodward Medical Research Institute, Buffalo, NY); letrozole was a gift from Novartis Pharma (Basel, Switzerland). Triptorelin pamoate was supplied by Ipsen (Milan, Italy). Solution of TAM was made in sesame oil; the other substances were dissolved daily in double-distilled water, and further dilutions to the final concentrations were made in Krebs’ solution.

Epididymal and corpora cavernosal tissue preparations Epididymis, corpus cavernosum, placenta, and uterus were obtained from New Zealand White rabbits. The animals were killed by a lethal dose of pentobarbital. Epididymis was carefully separated from testes and adherent fat. Tissue specimens were fresh frozen for RNA preparation and Western blot analysis. The penis was removed and the corpora cavernosa were carefully dissected free from the tunica albuginea. For in vitro contractility studies epididymis and corpus cavernosum (CC) preparations were immediately placed and maintained in cold Krebs solution until the beginning of the experiments.

In vitro contractility The epididymis, at the border between corpus and cauda, was cut into three to four small strips (0.5 ⫻ 0.3 ⫻ 0.1 cm). Rabbit strips were vertically mounted under 700-mg resting tension in organ chambers containing 10 ml Krebs solution at 37 C, gassed with 95% O2 and 5% CO2 at pH 7.4. The solution had the following composition: NaCl, 118 mm; KCl, 4.7 mm; KH2PO4, 1.2 mm; MgSO4, 1.2 mm; NaHCO3, 25 mm; CaCl2, 2.5 mm; and glucose, 10 mm. The preparations were allowed to equilibrate for at least 90 min; during this period the bath medium was replaced every 15 min. Changes in isometric tension were recorded on a chart polygraph (Battaglia Rangoni, San Giorgio di Piano, Bologna, Italy). NA (0.1–10 ␮m) increased the tonic tension in a concentrationdependent manner, with a maximum effect obtained at 10 ␮m. This value was taken as 100% and the increase recorded in the presence of different concentrations of OT (0.01–10,000 nm) and its analogs were referred to this value. Drug cumulative concentrations were added, at 7-min intervals, to the bath to obtain a concentration-dependent curve; a 30- to 60-min pretreatment with selected antagonists was performed before the concentration-response curve for the agonist. The CCs were cut into three to four strips (0.2 ⫻ 0.2 ⫻ 0.7 cm). Strips were vertically mounted under 1.8 g resting tension in organ chambers containing 10 ml Krebs solution with the same composition as described above, at 37 C, gassed with 95% O2 and 5% CO2 at pH 7.4. The preparations were allowed to equilibrate for at least 90 min; during this period the bath medium was replaced every 15 min. Phenylephrine (Phe, 0.1–100 ␮m) increased the tonic tension in a concentration-dependent manner, with a maximum effect obtained at 100 ␮m. This value was taken as 100%, and the increase recorded in the presence of different concentrations of ET-1 was referred to this value. Drug cumulative concentrations were added, at 7-min intervals, to the bath to obtain a concentration-dependent curve.

Experimental hypogonadism and sex steroid replacement The study was approved by the Local Ethical Committee for Investigations in Animals of the University of Florence. New Zealand White male rabbits (weighing approximately 2.5 kg, n ⫽ 36) were divided into four groups. One group was kept intact (controls, n ⫽ 9). Another group was treated with a single administration of 2.9 mg/kg of the long-acting GnRH analog triptorelin pamoate (n ⫽ 18) or vehicle (n ⫽ 3). After 15 d, a subset of GnRH-treated rabbits (n ⫽ 9) were supplemented with a pharmacological dose of testosterone enanthate (T, 30 mg/kg weekly, n ⫽ 3), estradiol (E2) valerate (E2v, 3.3 mg/kg, n ⫽ 3) or TAM (0.250

Filippi et al. • OT and Sex Steroids in Rabbit Epididymis

mg/kg, daily, n ⫽ 3). After 2 months from triptorelin pamoate administration and after 1 wk from the last supplementation of T/E2v or after 1 d from TAM, rabbits were killed and blood was drawn from the heart for sex steroid measurement. Another group of sexually mature, intact animals (weighing approximately 3 kg) was treated for 3 wk with letrozole (2.5 mg/kg, daily, n ⫽ 3), an aromatase inhibitor (29), dissolved in the drinking water or vehicle (n ⫽ 3). Experiments with letrozole and its relative control were carried out at springtime when the circulating levels of T reach their zenith (30).

Measurement of T and E2 Plasma level of T and E2 was measured with an automated chemiluminescence system (Bayer Corp. Diagnostics, East Walpole, MA) after appropriate extraction. For extraction, samples were mixed with 4 volumes of diethyl ester for 15 min, centrifuged for 5 min at 2000 rpm, and the aqueous phase frozen in dry ice. The organic phase was recovered, evaporated to dryness under a nitrogen stream, and reconstituted in the assay buffer.

RT-PCR Total RNA was extracted from rabbit epididymis with RNAase midi kit from QIAGEN (Valencia, CA). RNA concentrations were determined by spectrophotometric analysis at 260 nm. RT-PCR experiments were performed as previously reported (17). Briefly, total RNA (500 ng) was retrotranscribed for 30 min at 50 C, denatured for 2 min at 95 C, and amplified for 30 cycles with the following parameters: denaturation 45 sec at 95 C, annealing 1 min at 55 C, and extension 1 min at 70 C. The specific primers for rabbit OTR covered a 317-bp region of the rabbit OTR mRNA sequence, as deposited in the GenBank at NCBI (accession no. AF023851). The sequence of sense primer (position 110 –129) was 5⬘-ATG TTT GCC TCC ACC CAC AT-3⬘; the sequence of antisense primer (position 398 – 427) was 5⬘-CCA GAT CTT GAA GCT GAT GA-3⬘. The integrity of total RNA was verified performing the RT-PCR for the rabbit housekeeping ␥-nonmuscle actin (␥-ACT). The ␥-ACT-specific primers covered a 328-bp region of rabbit ␥-ACT sequence, as deposited in the gene GenBank at NCBI (accession no. X60733). The sequence of sense primer (position 317–336) was: 5⬘-ACA TGG AGA AGA TCT GGC AC-3⬘; the sequence of antisense primer (position 626 – 645) was: 5⬘-CAT GAG GTA GTC GGT CAG GT-3⬘. The amplified cDNA were fractionated on a 2% agarose gel, and the specific bands were excised from the gel and purified from agarose using the Concert gel extraction systems kit (Invitrogen, Milan, Italy). After purification, the amplified cDNAs were customer sequenced (MGW-Biotech, Florence, Italy) to confirm that their sequences corresponded to the rabbit OTR and ␥-ACT sequences as deposited in GenBank. The contamination of genomic DNA was excluded performing 35 cycles of amplification without retrotranscription.

Northern blot analysis Thirty micrograms total RNA were fractionated on a 1.2% agarose gel containing 8% formaldehyde. RNAs were then transferred onto nylon membrane (Hybond-n, Amersham, Milan, Italy) and baked at 80 C for 2 h. Membranes were prehybridized for 1 h and hybridized overnight at 65 C with Church & Gilbert buffer solution, as previously described (31). The probe for the detection of OTR mRNA was derived from RT-PCR of total epididymal rabbit RNA, as described in the previous section. The probe for the detection of rabbit clusterin was a specific rat clusterin 1.5-kb full-length cDNA (32). The probes were labeled with deoxycytidine 5⬘-[␣-32P] triphosphate by a random priming kit (Roche) and chromatographed (Nu-Clean D25 disposable spun columns, IBI, New Haven, CT) before use. The hybridized nylon membranes were submitted to autoradiography using Hyperfilm-MP (Amersham) and X-Omatic Regular intensifying screen (Kodak, Rochester, NY) at ⫺80 C for various exposition times.

SDS-PAGE and Western blot analysis All samples were split in two for RNA and protein analysis. For protein analysis, frozen samples were directly suspended in lysis buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 0.25% Nonidet P-40, 1 mm Na3VO4,


1 mm phenylmethylsulfonyl fluoride) and homogenized (Teflon-glass). The homogenates were centrifuged 2000 rpm for 10 min at 4 C, and the protein content of supernatants was evaluated according to the Bradford method using Coomassie (Bio-Rad Laboratories, Inc.). After protein measurement, aliquots containing 30 ␮g proteins were diluted in reducing 2 ⫻ Laemmli’s sample buffer (62.5 mm Tris, pH 6.8; 10% glycerol; 20% SDS; 2.5% pyronin; and 100 mm dithioteithrol) and loaded onto 10% SDS-PAGE. After separation by SDS-PAGE, proteins were transferred to nitrocellulose membranes. Membranes were blocked 2 h at room temperature in 5% milk-Tween Tris buffer saline (TTBS, 0.1% Tween-20, 20 mm Tris, 150 mm NaCl), washed in TTBS and incubated overnight with primary antibodies (in 5% milk-TTBS) followed by peroxidase-conjugated secondary IgG (1:3000). Finally, reacted proteins were revealed by a BM enhanced-chemiluminescence system.

Densitometric analysis Images of the gels and films were scanned with a plot bed scanner (IS440CF, Kodak Digital Science). Quantification of the bands was made using Photoshop 5.5 software (Adobe Systems Incorporated Italia srl Agrate Brianza, Milano, Italy).

Statistical analysis Results are expressed as mean ⫾ sem for n experiments. Statistical analysis was performed with t test for paired or unpaired data, with ANOVA followed by Fisher’s test to evaluate the differences among

FIG. 1. Characterization of OTR in the rabbit epididymis. A, Effect of increasing concentrations of OT (closed circles, n ⫽ 27, in 15 separate experiments), [Thr4, Gly7]OT (closed squares, n ⫽ 6, in three separate experiments), (Phe2, Orn8)-oxytocin (closeddown triangles, n ⫽ 4, in three separate experiments), and DDAVP (closed-up triangles, n ⫽ 5, in four separate experiments) on the basal tone of rabbit epididymal preparations. Ordinate: contractile activity, expressed as percentage of the maximal response obtained with NA (10 ␮M); abscissa: concentration of the agonists. Data were expressed as the means ⫾ SEM. B, Effect of increasing concentrations of (d(CH2)51 Tyr(Me)2Orn8)-oxytocin, a specific OT antagonist, on the tone induced by a fixed concentration of OT (1 ␮M) in the rabbit epididymis (open circles, n ⫽ 6 in four separate experiments). Relative IC50 and EC50 are reported in the text.

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groups, and P less than 0.05 was taken as significant. EC50 and IC50 values were calculated by the computer program ALLFIT (33).

Results

Figure 1A shows the effect of OT and [Thr4, Gly7]OT (a selective OTR agonist) on the contractility of rabbit epididymis. Both OT and [Thr4, Gly7]OT elicited a rather similar increase in epididymal tone (Emax ⫽ 75.5% ⫾ 4.0%) with EC50s ⫽ 221 ⫾ 80 nm and 147 ⫾ 35 nm, respectively. Conversely, (Phe2, Orn8)-oxytocin (a selective V1 vasopressin agonist) induced a less sustained contractility than OT (Emax ⫽ 33% ⫾ 1.8%, P ⬍ 0.05) with EC50 ⫽ 280 ⫾ 39.5 nm. The selective V2 vasopressin agonist DDAVP was almost ineffective. The stimulatory effect of OT (1 ␮m) was almost completely counteracted by the selective OT antagonist (d(CH2)51 Tyr(Me)2Orn8)-oxytocin (Imax ⫽ 67% ⫾ 10%, IC50 ⫽ 31.9 ⫾ 23 nm, Fig. 1B). These findings indicate that OTR is present in rabbit epididymis and OTR mediates the majority of the contractile effect of OT. To verify whether the responsiveness to OT is somehow affected by changing the endocrine milieu, we treated adult rabbits with the long-acting GnRH agonist triptorelin (2.9

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mg/kg). This treatment was designed to obtain a pharmacological model of hypogonadotropic hypogonadism by reducing the hypophyseal stimulation to the testis but leaving the anatomic and metabolic connections between the male gonad and epididymis intact. A subset of rabbits was supplemented, after 2 wk, with T enanthate (30 mg/kg). Table 1 shows the level of T in the peripheral blood at the time of the experiment (2 months after GnRH analog administration TABLE 1. Effect of a GnRH analog (triptorelin) with or without T replacement on sex steroid plasma levels

a) Control (n ⫽ 3) b) Vehicle (n ⫽ 3) c) Triptorelin (n ⫽ 3) d) Triptorelin ⫹ T (n ⫽ 3)

T (nmol/liter)

E2 (pmol/liter)

4.54 ⫾ 0.90 5.73 ⫾ 1.40 0.60 ⫾ 0.10a 2.30 ⫾ 0.40

58.3 ⫾ 4.70 60.5 ⫾ 6.50 24.9 ⫾ 4.93a 45.3 ⫾ 17.70

Blood for T and E2 measurement was drawn after 2 months from a single administration of the long-acting GnRH analog triptorelin pamoate (2.9 mg/kg d 1, groups c and d) or vehicle (group b) and after 1 wk from the last injection of T enanthate (30 mg/kg 䡠 wk from d 14, group d). Triptorelin pamoate significantly reduced T and E2 plasma levels in group c. Weekly injection with the long-lasting T ester (d) restored T and E2 plasma levels to values not significantly different from untreated (a) or vehicle-treated rabbits (b). a P ⬍ 0.01 vs. a, b, and d. n, Number of animals.

and 1 wk after the last administration of T or vehicle). Triptorelin administration significantly decreased T plasma levels, compared with controls or vehicle-treated rabbits (P ⬍ 0.01). Supplementation of triptorelin-treated rabbits with T enanthate restored T concentration to the levels observed in controls and vehicle-treated rabbits. Figure 2A shows the responsiveness of epididymis to OT in the different groups of animals. Chronic treatment with the GnRH agonist almost abolished epididymal sensitivity to OT (P ⬍ 0.01 vs. control and vehicle). Surprisingly, T supplementation did not restore responsiveness to OT. To verify whether the lack of responsiveness to OT in the T-treated rabbits was due to an insufficient degree of androgenization, we studied another androgen-dependent tissue from the same rabbits, CC. In CC we studied the responsiveness to a well-known contractile agent, ET-1. Results are shown in Fig. 2B. Increasing concentrations of ET-1 induced a dose-dependent increase in the tension of CCs from both controls and vehicle-treated rabbits. As observed in epididymis, triptorelin administration significantly blunted the sensitivity of CC to ET-1. However, in this tissue, supplementation with T not only completely rescued ET-1 responsiveness but also even increased the sensitivity to ET-1 over both controls and

FIG. 2. Effect of GnRH-induced hypogonadism and T replacement on responsiveness to OT (epididymis) and ET-1 (CC). A, Effect of increasing concentrations of OT on the basal tone of epididymal preparations in untreated (closed circles, n ⫽ 5 in three separate experiments), vehicle-treated (open circles, n ⫽ 6 in three separate experiments), and hypogonadal rabbits supplemented with (closed triangles, n ⫽ 6 in three separate experiments) or without (closed squares, n ⫽ 5 in three separate experiments) weekly administration of T enanthate. Ordinate: contractile activity, expressed as percentage of the maximal response obtained with NA (10 ␮M); abscissa: concentration of oxytocin. Data were expressed as the means ⫾ SEM. **, P ⬍ 0.01 vs. untreated (closed circles) and vehicle-treated (open circles). B, Effect of increasing concentrations of ET-1 on the basal tone of CC preparations in untreated (closed circles, n ⫽ 5 in three separate experiments), vehicle-treated (open circles, n ⫽ 5 in three separate experiments), and hypogonadal rabbit with (closed triangles, n ⫽ 6 in three separate experiments) or without (closed squares, n ⫽ 7 in three separate experiments) weekly administration of T enanthate. Ordinate: contractile activity, expressed as percentage of the maximal response obtained with Phe (100 ␮M); abscissa: concentration of ET-1. Data were expressed as the means ⫾ SEM. *, P ⬍ 0.05 vs. untreated (closed circles) and vehicle treated (open circles). **, P ⬍ 0.01 vs. untreated (closed circles) and vehicle treated (open circles).


vehicle-treated rabbits (P ⬍ 0.01). Hence, the pharmacological treatment with T enanthate was more than sufficient to restore ET-1 sensitivity in CC but not OT sensitivity in the epididymis of hypogonadal rabbits. We therefore hypothesized that other factors secreted by the testis might control OT responsiveness in epididymis. T is not the only steroid produced by the testis under gonadotropin control. Estrogens are also produced and secreted by Leydig cells. Indeed, the peripheral concentration of E2 was significantly decreased in triptorelin-treated rabbits (Table 1). Because estrogen receptor (ER) has been described in the epididymis of various animal species, we tested the possibility that rabbit epididymis expresses one or both subtypes of the ER, i.e. ER␣ and ER␤. Figure 3A shows a Western blot analysis of the three portions of rabbit epididymis and placenta (positive control) obtained using the monoclonal rat H222 antibody that recognizes both subtypes of ER (34). As shown, specific bands for ER␣ and ER␤ are present in the placenta as well as in the different portions of rabbit epididymis. This indicates that rabbit epididymis is a possible target for estrogens. Moreover aromatase is present in all the three portions of epididymis (caput, corpus, and cauda) as demonstrated by Western blot analysis of lysates obtained from the three regions, revealed by a specific antibody. Although T enanthate administration reverted the GnRH-induced E2 decrease in peripheral blood because of its aromatization to estrogens (Table 1), we hy-


pothesized that the T treatment did not restore the local (epididymal) concentration of estrogens, which are known to be several-fold higher than in the peripheral blood (35). We therefore repeated the experiment with the long-acting GnRH agonist but substituting, as hormonal replacement, E2v (3.3 mg/kg) to T enanthate. As expected, E2v treatment did not restored the GnRH-induced fall in T plasma levels, but dramatically increased the peripheral concentrations of E2 (Table 2). The effect of this pharmacological rise in estrogen plasma levels is shown in Fig. 4. Epididymal responsiveness to OT was blunted by triptorelin, as previously observed. The pharmacological administration of estrogens to GnRH-treated rabbits not only restored but even amplified the sensitivity of epididymis to OT (Fig. 4A) by increasing over the control both the EC50 (EC50 ⫽ 2.2 ⫾ 0.5 nm, P ⬍ 0.01) and the Emax (Emax ⫽ 100.6 ⫾ 3.1, P ⬍ 0.01). Conversely, in the CCs of the same rabbits, the ET-1 responsiveness, blunted by triptorelin administration, was not affected by the pharmacological treatment with E2v (Fig. 4B). These results, taken together, strongly indicate that the hyporesponsiveness to OT in epididymis and to ET-1 in CC induced by hypogonadism was restored by different hormonal treatment with sex steroids: estrogens for the former and androgens for the latter. To further investigate on the effect of estrogens on OT responsiveness in epididymis, we repeated the aforementioned experiments with triptorelin, treating the hypogonadal rabbit with the ER antagonist TAM (0.25 mg/kg daily, after 2 wk from triptorelin administration). TAM administration did not affected E2 plasma levels (53.3 ⫾ 3.3 pmol/liter) but restored and even amplified OT sensitivity in epididymis (Fig. 5). This finding indicates that TAM acts on epididymis not as an ER antagonist but as an ER agonist, as previously described on uterus (36). Indeed, TAM is now considered more a selective estrogen receptor modulator than an ER antagonist. Although all the previous evidence indicates that a pharmacological treatment with estrogens regulates OT sensitivity in epididymis, they do not support a role for endogenous estrogens in modulating OT responsiveness. We therefore employed a different pharmacological model. We treated sexually mature, intact rabbits for 3 wk with a potent and selective aromatase inhibitor, letrozole (29, 2.5 mg/kg, daily). Letrozole administration induced a 66.6% ⫾ 10.2% decrease in E2 plasma levels in treated rabbits (P ⬍ 0.01). This decline TABLE 2. Effect of a GnRH analog (triptorelin) with or without E2 replacement on sex steroid plasma levels

FIG. 3. Western blot detection of ER␣ and ER␤ and aromatase in rabbit epididymis. Thirty micrograms proteins obtained from different regions of rabbit epididymis and from rabbit placenta were separated by 10% SDS-PAGE, transferred onto nitrocellulose membrane, and probed for ER expression with rat monoclonal H222 antibody. A, Two bands of about 65 and 55 kDa (arrowheads) corresponding, respectively, to ER␣ and ER␤ are present in both the different portions of rabbit epididymis and placenta, used as positive control. B, The same nitrocellulose was stripped and reprobed for aromatase using a rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase. A single band at the expected molecular mass is present in both the different portions of rabbit epididymis and placenta, used as positive control. Molecular-mass markers (kilodaltons) are indicated on the left of the blot.

a) Control (n ⫽ 3) b) Triptorelin (n ⫽ 3) c) Triptorelin ⫹ E2 (n ⫽ 3)

T (nmol/liter)

E2 (pmol/liter)

5.05 ⫾ 1.00 1.02 ⫾ 0.20a 2.00 ⫾ 0.20a

50.20 ⫾ 6.20 27.30 ⫾ 5.55a 1190 ⫾ 635.80a

Blood for T and E2 measurement was drawn after 2 months from a single administration of the long-acting GnRH analog triptorelin pamoate (2.9 mg/kg d 1, groups b and c) and after 1 wk from the last injection of E2 valerate (3.3 mg/kg 䡠 wk from d 14, group c). Triptorelin pamoate significantly reduced T and E2 plasma levels in group b. Weekly injection with the long-lasting E2v did not restore T plasma levels to values not significantly different from untreated animals (a), whereas it strongly enhanced E2 plasma levels in group c. a P ⬍ 0.01 vs. a. n, Number of animals.

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FIG. 4. Effect of hypogonadism and E2 replacement. A, Effect of increasing concentrations of OT on the basal tone of epididymal preparations in untreated (closed circles, n ⫽ 5, in three separate experiments) and hypogonadal rabbit with (closed triangles, n ⫽ 6 in three separate experiments) or without (closed squares, n ⫽ 8 in three separate experiments) weekly administration of E2v. Ordinate: contractile activity, expressed as percentage of the maximal response obtained with NA (10 ␮M); abscissa: concentration of oxytocin. Data were expressed as the means ⫾ SEM. **, P ⬍ 0.01 vs. untreated (closed circles). B, Effect of increasing concentrations of ET-1 on the basal tone of CC preparations in untreated (closed circles, n ⫽ 5 in three separate experiments) and hypogonadal rabbit with (closed triangles, n ⫽ 5 in three separate experiments) or without (closed squares, n ⫽ 5 in three separate experiments) weekly administration of E2v. Ordinate: contractile activity, expressed as percentage of the maximal response obtained with Phe (100 ␮M); abscissa: concentration of ET-1. Data were expressed as the means ⫾ SEM. **, P ⬍ 0.01 vs. untreated (closed circles).

in endogenous estrogens induced by letrozole corresponded to a loss in epididymal sensitivity to OT, similar to that observed in hypogonadal rabbits (Fig. 6). Therefore, we demonstrated that both exogenous and endogenous estrogens regulate epididymal responsiveness to OT. To investigate whether the estrogen-induced increased responsiveness to OT was related to an up-regulation of OTR, as previously described in the uterus and mammary gland (37), we studied OTR gene and protein expression in the epididymis of hypogonadal rabbits replaced or not with sex steroids. OTR mRNA present in epididymis was characterized by Northern analysis and RT-PCR (Fig. 7, A and B). Epididymis expresses two major RNA bands that hybridized to a rabbit OTR cDNA probe. These two bands were of the same size of those found in rabbit uterus and were estimated at 2.7 and 3.7 kb. A third OTR mRNA band of 1.3 kb expressed in the uterus was barely detectable in the epididymis. As a control, the same blot was rehybridized with a clusterin-specific cDNA probe (Fig. 7A). E2, but not T, administration increases the OTR mRNA abundance, and both sex steroids down-regulate the castration-induced clusterin mRNA expression. By using specific RT-PCR primer pairs for rabbit OTR, we obtained in the epididymis a single band that was identical with the one obtained with uterine RNA and corresponded to the size that was predicted from the rabbit OTR gene sequence (Fig 7B). Again, E2, but not T, increases the abundance of the OTR transcript in epididymis. Similar results were obtained by Western analysis on OTR protein

expression by using a specific antibody. We detected a single band of 55 kDa in uterus, used as positive control, and in epididymis: This band was up-regulated by E2 but not by T (Fig. 7C). The semiquantitative evaluation of OTR mRNA abundance, shown in Fig. 7D, is based on the densitometric analysis of the most prominent 2.7-kb transcript, as detected in Northern analysis and from the analysis of the 317-bp fragment by RT-PCR, after correction for the 18s and ␥-ACT expressions, respectively. The semiquantitative evaluation of the 55-kDa band detected by Western analysis is also represented (Fig. 7D). The extent of the E2-induced OTR increase was rather similar in both OTR protein and gene expression (Fig. 7D). Discussion

This study demonstrates the presence of functionally active OTR in rabbit epididymis and its regulation by estrogens. The presence of OTR in epididymis is not a new finding; indeed, its presence has been postulated for a long time (21–23) and recently demonstrated in several animal species (18 –20, 38), including humans (17, 20). In this study, by using selective agonist and antagonist of neurohypophyseal hormone receptors, we showed that OTR is involved in promoting epididymal contractility, confirming previous preliminary results (17, 23). Stimulation by OT of epididymal motility might be important in promoting the release of sperm stored at this level and therefore allowing semen


FIG. 5. Effect of hypogonadism and TAM replacement. Effect of increasing concentrations of OT on the basal tone of epididymal preparations in untreated (closed circles, n ⫽ 6 in three separate experiments) and hypogonadal rabbit with (closed triangles, n ⫽ 5 in three separate experiments) or without (closed squares, n ⫽ 6 in three separate experiments) daily administration of TAM (0.25 mg/kg) Ordinate: contractile activity, expressed as percentage of the maximal response obtained with NA (10 ␮M); abscissa: concentration of oxytocin. Data were expressed as the means ⫾ SEM. *, P ⬍ 0.05 and **, P ⬍ 0.01 vs. untreated (closed circles).

emission. In fact, it was known that OT administration increased the number of sperm ejaculated (9 –17, 38) and that this might have a physiological role because circulating OT increases at the time of ejaculation (4 – 8). Besides the demonstration of functional OTR in epididymis, the most relevant findings in our study are: 1) ER␣ and ER␤ are expressed by rabbit epididymis together with aromatase; 2) the effect of OT on epididymal motility is regulated by estrogens; and 3) this is at least in part caused by an estrogen-mediated up-regulation of OTR gene and protein. The presence of ER in epididymis was previously demonstrated in several mammals including rabbit (39, 40) and human (26). Because high concentrations of estrogens of testicular origin are present in the rete testis and epididymis (41), it was supposed that ER in the epididymis plays some physiological roles. This was later clearly demonstrated in a mouse model exhibiting a functional knockout of ER␣ (42, 43). In fact, mice genetically lacking ER␣ are subfertile, having important defects in the fluid reabsorption in the epididymis (44) because of an impaired synthesis of several proteins including the sodium/hydrogen exchanger-3 (45). Moreover, the high level of T in the epididymis and the concomitant presence of aromatase in all the three portions of epididymis strongly suggest that epididymis can actively metabolize T in E2, further increasing estrogen concentration at this level. In addition, aromatase activity is also present in germ cells (46) and therefore sperms stored in the epididymis might represent an additional source of local estrogen (47). We now clearly demonstrated an additional action of estrogens in epididymis: up-regulation of OT responsiveness.


FIG. 6. Effect of aromatase inhibition in sexually mature intact rabbits. Effect of increasing concentrations of OT on the basal tone of epididymal preparations in sexually mature intact rabbits untreated (closed circles, n ⫽ 6 in three separate experiments) or treated with letrozole (closed triangles, n ⫽ 6 in three separate experiments). Ordinate: contractile activity, expressed as percentage of the maximal response obtained with NA (10 ␮M); abscissa: concentration of oxytocin. Data were expressed as the means ⫾ SEM. *, P ⬍ 0.05 and **, P ⬍ 0.01 vs. untreated (closed circles).

In fact, in a pharmacological model of hypogonadotropic hypogonadism in rabbit estrogens (induced by a long-acting GnRH agonist), but not androgens, fully restored sensitivity to OT. This was peculiar of OT responsiveness in epididymis because ET-1 sensitivity in the penis of the same rabbits showed the opposite regulation: restored by androgens, but not by estrogens. In addition, deprivation of endogenous estrogens, by blocking their formation using an aromatase inhibitor, induced OT hyporesponsiveness comparable with that observed in hypogonadic rabbits. Therefore, estrogens, most probably derived from the testis and epididymal T aromatization, act on ER in the epididymis, increasing the sensitivity to OT. Because in several tissues, including uterus (48 –51), cervix (52), hypothalamus (53), kidney (54), and blood vessels (55), the OTR gene and protein are up-regulated by estrogens, we tested whether this was the case also in epididymis. We found that chronic administration of E2v to hypogonadal rabbits induced a 2-fold increase in epididymal expression of OTR gene transcript and immunoreactivity, but it reduced the castration-induced rise of a sex steroid-dependent gene as clusterin. The mechanism of action of estrogens on OTR remains unknown at the present time. Although OTR promoter contains a palindromic ER element and several half-ER elements, it appears that these promoter elements were nonfunctional (56). Hence, it is possible that the positive effect of estrogens on OTR is not directly transcriptionally regulated but mediated by still unknown indirect mechanisms. Interestingly, an ER antagonist, TAM, did not suppress but even stimulated OT responsiveness in epididymis of hypogonadal rabbits. However, it is

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FIG. 7. OTR expression in rabbit epididymis. A, Expression of OTR and clusterin genes (upper and lower blots, respectively) as detected in a typical experiment of Northern analysis. Each lane was loaded with 30 ␮g total epididymis RNA. The blot was hybridized with an OTR-specific cDNA probe stripped and thereafter reprobed with a clusterin-specific cDNA probe. The probes were labeled with 32P. The calculated size (kilobase pairs) of the specific transcript is indicated on the right. Total RNA extracted from rabbit uterus was used as positive control. Corresponding ethidium bromide staining of the gel is shown below the blot. B, RT-PCR products from total epididymis RNA using OTR- (upper), and ␥-ACT-specific (lower) primers. Rabbit uterus total RNA was used as positive control. ␥-ACT mRNA amplification was used as housekeeping gene. MWM, Molecular-mass marker. C, Western blot detection of OTR protein in rabbit epididymis. Thirty micrograms proteins obtained from epididymis of differently treated rabbits and from rabbit uterus were separated by 10% SDS-PAGE, transferred onto nitrocellulose membrane, and probed for OTR expression with monoclonal 3–12 antibody (5 ␮g/ml antibody). A single band of about 55-kDa (arrow) is present in both epididymal samples and uterus, used as positive control. Molecular-mass markers (kilodaltons) are indicated on the right of the blot. D, Mean ⫾ SEM of the percentage increase over Triptorelin, taken as 100%, of OTR gene (empty bars) and protein (filled bars) expression in the indicated number of experiments. Quantification of OTR band intensity was made directly on the film (Northern and Western) or on the gel (RT-PCR) by image scanning analysis. *, P ⬍ 0.05 vs. Triptorelin.

well known that TAM has tissue-specific estrogenic effects, acting as a selective estrogen receptor modulator. An estrogen-like effect of TAM in rabbit epididymis was previously reported (57). Hence, it is possible that in rabbit epididymis, as well as in endometrium (36), TAM retains estrogenic activity. This OT-sensitizing activity of TAM might underlie

some positive effect of this drug on male reproduction. In fact, TAM is still recommended for the treatment of male subfertility, and in particular for oligozoospermia (58). It was hypothesized that TAM acting as an estrogen antagonist at the hypothalamic/pituitary level stimulates gonadotrophin secretion and therefore spermatogenesis (59). We now sug-


gest that in addition to its central estrogen antagonistic properties, TAM behaves as an estrogen agonist at the level of epididymis. Our results on the estrogen regulation of OTR responsiveness are an additional contribution on the new notion that estrogens are necessary for not only female reproduction but also male reproduction. Acknowledgments We thank Paolo Ceccatelli and Mauro Beni (CESAL, University of Florence) for technical assistance in animal treatment; Dr. T. Kimura (Department of Obstetrics and Gynecology, Osaka, Japan), Prof. G. Greene (University of Chicago) and Dr. C. Yarborough (HauptmanWoodward Medical Research Institute, Buffalo, NY) for kindly providing the anti-OTR monoclonal antibody 3–12, monoclonal rat H222 antibody, and antiaromatase polyclonal antibody, respectively; Prof. S. Ando` (University of Calabria, Italy) for his helpful suggestions and comments. Received April 5, 2002. Accepted July 24, 2002. Address all correspondence and requests for reprints to: Professor Mario Maggi, Department of Clinical Physiopathology, University of Florence, V.le G. Pieraccini, 6, 50139 Florence Italy. E-mail: m.maggi@ dfc.unifi.it. This study was supported by a grant from the University of Florence, Florence, Italy.

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