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BOR - Papers in Press, published online ahead of print October 17, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.009209
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BIOLOGY OF REPRODUCTION 68, 588–594 (2003)
DOI: 10.1095/biolreprod.102.009209
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Quantification of Prolactin (PRL) and PRL Receptor Messenger RNA in Gilthead Seabream (Sparus aurata) after Treatment with Estradiol-17ß1

J. Eduardo B. Cavacoa, Cecília R.A. Santosa, Patricia M. Ingletonb, Adelino V.M. Canarioa, and Deborah M. Power2,a

a Centro de Ciências do MAR (CCMAR), Universidade do Algarve, Campus de Gambelas, 8000-117 Faro, Portugal b Academic Unit of Endocrinology, Division of Genomic Medicine, Medical School, Sheffield S102 RX, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prolactin (PRL) in fish is considered to be an osmoregulatory hormone, although some studies suggest that it may influence the production of steroid hormones in the gonads. The objective of the present study was to establish if PRL is involved in reproduction of the gilthead seabream—a protandrous hermaphrodite. Adult and juvenile gilthead seabream received implants of estradiol-17ß (E2) for 1 wk during the breeding season, and the mRNA expressions of PRL and PRL receptor (sbPRLR) were determined. Northern blot analysis revealed a single pituitary PRL transcript, the expression of which was significantly reduced by E2 treatment in adults but significantly increased in juvenile fish. In adult gonads, four sbPRLR transcripts of 1.1, 1.3, 1.9, and 2.8 kilobases were observed. A competitive reverse transcription-polymerase chain reaction was developed and used to determine how E2 treatment alters expression of the gonadal sbPRLR gene. Seabream PRLR was detectable in all samples analyzed by this assay. Levels of sbPRLR mRNA increased significantly (50-fold) after E2 treatment in adults, but a 24-fold decrease was measured in juveniles. Immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR detected the receptor in spermatogonia and oocytes. Taken together, the preceding results suggest that in the seabream, PRL may act on both testis and ovary via its receptor and that the stage of maturity influences this process. The full characterization and relative importance of the different transcripts of sbPRLR in eliciting the action of PRL in the gonads remain to be elucidated.

estradiol, pituitary, prolactin, prolactin receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prolactin (PRL) is a 23-kDa polypeptidic hormone secreted by the anterior pituitary in all vertebrates [1, 2]. The actions of PRL are mediated through a single-pass transmembrane receptor, the PRL receptor (PRLR), which belongs to the class 1 cytokine receptor superfamily [3]. Associations of PRL with PRLR trigger the postreceptor signaling events, which culminate in a large number of physiological actions (>300 have been identified) that can be divided into the following categories: water and electrolyte balance, growth and development, endocrine and metabolism, brain and behavior, reproduction, and immunoregulation and protection (for review, see [4]). One of the well-known actions of PRL is in the reproductive physiology of mammals. For example, PRL acts on the mammary gland and specifically stimulates DNA synthesis, epithelial cell proliferation, and synthesis of milk proteins (casein and lactalbumin), free fatty acids, and lactose [5]. Prolactin has also been shown to regulate luteal functions, follicular steroidogenesis, and follicular growth in the ovary during reproduction [6, 7], and homozygous female PRLR knockout mice are completely infertile and lack normal mammary gland development [8]. In male animals, PRL plays an important role in maturation of the gonads, and PRL supplementation to immature, hypophysectomized rats stimulates the multiplication and differentiation of Leydig cells and germ cells in a dose-dependent manner [9]. Barkey et al. [10] and Hondo et al. [11] have shown that PRLRs are expressed in Leydig cells, Sertoli cells, and spermatogonia. These observations suggest that in the testis, PRL may be a trophic hormone that acts directly on testicular tissue.

In fish, PRL is considered to be primarily an osmoregulatory hormone [12], although some studies suggest PRL may be associated with production of steroid hormones in the gonads, the onset of gonadal development, and reproductive behavior [13]. Mammalian PRL stimulated testosterone production in the goby (Gobius niger) [14] suggesting a possible role for PRL in the regulation of testicular function. Furthermore, specific PRL-binding sites have been detected in seminal vesicle cells of the same species [15] and in tilapia (Oreochromis mossambicus) testis [16].

Recently, PRL and PRLR have been cloned from the gilthead seabream (Sparus aurata) [17, 18]. A single transcript of pituitary PRL was found encoding 188 amino acids, and studies using in situ hybridization demonstrated its abundant localization (25% of total volume of the pituitary) in the PRL cells of the rostral pars distalis [17]. The cloned seabream PRLR (sbPRLR) exhibits all the characteristic features of long forms of PRLRs and is expressed in many tissues, particularly the intestine, kidney, and gills [18].

In mammals, estrogens have a positive effect on PRL secretion. In addition to inducing hypertrophy of the lactotropic cells, estradiol (E2) increases PRL production by directly stimulating PRL gene transcription, leading to increased synthesis of PRL mRNA and PRL [19]. In teleosts, the way in which E2 influences PRLR expression in the gonads and the cellular localization of the receptor in fish have, to our knowledge, never been described. Thus, the primary aim of the present study was to determine the effects of E2 on the expression of PRL in the pituitary gland and on the gonadal expression of sbPRLR in the gilthead seabream. The gonads of this protandrous hermaphrodite are characterized by their bisexuality and consist of a mediodorsal ovarian area and a lateroventral testicular zone, separated by connective tissue [20]. During the first reproductive cycle, the ventral testicular part of the gonad proliferates and forms a mature testis, and from the second year onward, approximately 80% of the population undergo sex reversal and become functional females [20, 21]. The hermaphroditic nature and the poorly understood mechanisms underlying sex reversal of this species make the seabream an interesting comparative model for studying the involvement of PRL and its receptor in reproduction.

The generally low level of receptor expression means that methods such as mRNA blots, image analysis of in situ hybridization, and immunohistochemistry may be too insensitive for quantifying receptor gene expression. A sensitive competitive reverse transcription-polymerase chain reaction (C-RT-PCR) specific for the sbPRLR has therefore been developed. The cellular localization of sbPRLR in the gonads was determined by immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals, Experiments, Hormone Treatment, Sampling, and Radioimmunoassays

Adult and juvenile seabream obtained from outdoor ponds (Aquamarim; Aquacultura de Marim, Lda, Tavira, Portugal) were maintained at the Ramalhete Marine Station (Faro, Portugal). Experiments were carried out in 1000-L tanks receiving a constant flow of oxygenated seawater at 14 ± 2°C, under natural photoperiod for the Algarve-Portugal (November). All procedures involving experimental animals were conducted in accordance with Portuguese national regulations.

E2 treatment of adults Two-year-old gilthead seabream were treated with E2 implants in coconut oil (n = 12; 10 mg/kg body weight; Sigma-Aldrich, St. Louis, MO) when fish were in spermiation and the gonads contained more than 95% (w/w) testicular tissue. Control fish (n = 12) received similar, steroid-free implants. The length of the control and E2 fish were, respectively, 28.8 ± 0.28 cm and 28.5 ± 0.44 cm. One week after implantation, fish were anesthetized in 0.2% (v/v) aqueous phenoxyethanol (Sigma-Aldrich). Fish were weighed and measured, and blood samples were collected in 2-ml, heparinized syringes from the caudal vasculature. Plasma was stored at -20°C for future quantification of E2 levels by radioimmunoassay [22]. Fish were decapitated, and pituitaries were immediately dissected out and stored at -80°C until use. The gonads were removed and weighed, and the ratio between testicular tissue (white color) and ovarian tissue (yellow color) was scored. The gonadosomatic index (GSI) was calculated as gonad weight x 100/body weight. Subsequently, one of the gonads was immediately frozen in liquid nitrogen and stored at -80°C until molecular analysis; the other was fixed in Bouin-Holland fluid for histological and immunohistochemical analysis. The antiserum against the extracellular domain of sbPRLR has previously been characterized for immunohistochemistry [18].

E2 treatment of juveniles Juvenile gilthead seabream (n = 12 per group) were subjected to the same procedure outlined above for adults, except that the small size of the gonads did not allow for calculation of the ratio between testicular and ovarian tissue. The length of the control and E2 fish were, respectively, 18.4 ± 0.6 cm and 17.9 ± 0.5 cm. Only pituitaries and gonads were collected for quantification of pituitary PRL and gonadal sbPRLR expression.

Total RNA Extraction, mRNA Purification, and Northern Blot Analysis

Total RNA was extracted from whole pituitaries, from 500 mg of the gonads (adults), and from all gonads (juveniles) using TRI Reagent (Sigma-Aldrich) according to the manufacturer's protocol. Starting with 1.2 mg of total RNA from adult gonads, the poly(A)+ RNA fraction was obtained by chromatography on oligo(dT) cellulose columns (mRNA Purification Kit; Amersham-Pharmacia, Litle Chalfont, U.K.).

Pituitary total RNA (~5 µg for E2 treatment of adults and ~2 µg for E2 treatment of juveniles) and gonad poly(A)+ RNA (10 µg) were fractionated on a 5.5% (v/v) formaldehyde/1.5% (w/v) agarose gel and transferred to a Hybond-N (Amersham-Pharmacia) with 10x SSC (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate) and cross-linked with ultraviolet (UV) light. Before hybridization, the filter was washed at 60°C for 20 min in 1x SSC and 0.1% (v/v) SDS and prehybridized in 50% formamide, 50 mM sodium phosphate, 5x Denhardt solution, 0.1% SDS, 5x SSC, and 50 µg/ml of calf thymus DNA for 4 h at 42°C. Hybridization proceeded overnight at 42°C in fresh prehybridization solution containing probe radiolabeled with [{alpha}-32P]dCTP (NEN, Zaventem, Belgium) using random priming (Redi-Prime; Amersham-Pharmacia). The Northern blot with pituitary total RNA was probed with an {alpha}-32P-labeled dCTP full-length PRL cDNA; 10 µg of mRNA from gonads and 5 µg of total RNA from pituitary were probed with {alpha}-32P-labeled dCTP cDNA corresponding to the extracellular, transmembrane, and part of the intracellular domain of sbPRLR [18]. Filters were then washed for 30 min at 42°C in prehybridization solution, and stringency washes were carried out at 55°C for 15 min in 1x SSC and 0.1% SDS and then in 1x SSC and 0.1% SDS at 60°C for 30 min. The membranes were exposed to Biomax-MX film (Eastman Kodak, Rochester, NY) for 1 h (PRL and ß-actin) and 1 wk (sbPRLR) at -80°C. To evaluate the relative amounts of mRNA loaded in each sample, the membrane was hybridized with a gilthead seabream ß-actin probe [23] using the same hybridization protocol outlined above.

Competitive RT-PCR

Primer design for preparation of competitor A synthetic competitor fragment was prepared using the Competitive DNA Construction Kit (Takara, Tokyo, Japan). Briefly, specific primers for amplification of the target DNA were designed using the sbPRLR sequence aided by Primer Premier software (version 4.04; Premier Biosoft International, Palo Alto, CA): sense primer, 5'-AGTCCGGCTGGGTCACCATTA-3' (extracellular domain); and antisense primer, 5'-GGTGGCGACCAAGATCCAAAAC-3' (transmembrane domain). The expected product was 249 base pairs (bp). Primers were then prepared for competitor template DNA. Sense and antisense primers were composed of a sequence specific for the competitor template and were flanked by the sequence of sense and antisense primers of target DNA, respectively. In addition, the sense primer for the competitor template was flanked with the sequence of the SP6 promoter region. Primers were synthesized by MWG-Biotech GmbH (Ebersberg, Germany). The size of the DNA competitor was 369 bp.

Purification of DNA competitor The PCR was performed using the protocol and conditions suggested by the manufacturer, except that 1 µl of sense/antisense primer instead of 0.5 µl was used in the reaction. The PCR was carried out in a Robocycler (Stratagene, La Jolla, CA). Confirmation that the PCR product was of the expected size was obtained by running 2 µl of it on a 1.5% agarose gel (Gibco BRL, Barcelona, Spain) stained with ethidium bromide and visualized using UV illumination. Subsequently, residual primers and reagents were removed using GFx PCR DNA gel and band purification kit (Amersham-Pharmacia), and the purified DNA competitor was suspended in Tris-EDTA buffer.

Preparation of RNA competitor, purification, and quantification In vitro transcription was performed according to a standard protocol from Promega (Madison, WI) with a few modifications. Briefly, the reaction was carried out in a 20-µl reaction containing 200 mM Tris-HCl (pH 7.5), 30 mM MgCl2, 10 mM spermidine, 50 mM NaCl, 2 µl of 100 mM dithiothreitol (DTT), 1 µl (25 U) of RNA guard (Amersham-Pharmacia), 4 µl of each 2.5 mM ribonucleoside triphosphate, 7 µl of sterile water (Sigma-Aldrich), 1 µl of purified template DNA (see above), and 20 U/µl of SP6 Polymerase (Promega). The reaction was incubated for 2 h at 37°C. Subsequently, 2 µl of DNase (20 U; Amersham-Pharmacia) were added, and the reaction was incubated for 15 min at 37°C, followed by extraction with one volume of acid phenol:chloroform:isoamyl alcohol (25:24:1 [v/v/v], pH 4.5), vortexing for 1 min, and centrifugation at 12 000 x g for 2 min. The aqueous phase was transferred to a fresh tube, and one volume of chloroform:isoamyl alcohol (24:1 [v/v]) was added, after which it was vortexed and centrifuged at 12 000 x g for 2 min. The aqueous phase was then transferred to a fresh tube, and the cRNA was precipitated by addition of 2.2 µl of 3 M ammonium acetate and 55 µl of 100% ethanol and overnight incubation at -80°C. After centrifugation (12 000 x g for 10 min), the pellet was washed twice with 1 ml of 70% (v/v) ethanol, dried at room temperature, and resuspended in 50 µl of sterile distilled water. The cRNA concentration was determined by spectrophotometry (Gene Quant; Amersham-Pharmacia), and a 10-fold dilution of the cRNA competitor was prepared using RNase Free Glycogen (10 µg/ml; Takara), aliquoted, and stored at -80°C as standards for C-RT-PCR assays.

Validation of C-RT-PCR To confirm that the amplification efficiencies for competitor and target templates were similar, equal amounts of competitor cRNA and target RNA were introduced into the RT reaction, and synthesis was subsequently analyzed. The cDNA was synthesized from 3 µg of total RNA intestine (high abundance of sbPRLR [18]) plus 3 µg of competitor cRNA in a 40-µl reaction containing 0.05 M Tris-HCl (pH 8.3), 0.075 M KCl, 3 mM MgCl2, 2 µl of 0.1 M DTT, 10 mM of each dNTP, 50 pmol of antisense primer (sbPRLR), 5 U of RNA guard (Amersham-Pharmacia), and 40 U of Moloney murine leukemia virus reverse transcriptase (Gibco BRL) for 1 h at 37°C. The PCR was carried out in a 50-µl reaction containing 2 µl of the synthesized cDNA, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% (v/v) Triton X-100, 1.5 mM MgCl2, 5 mM of each dNTP, 25 pmol of sense and antisense primer (see above for sequences), and 1.25 U of Taq DNA polymerase (Promega). As a negative control, sterile water substituted for cDNA, and as positive controls, cDNA synthesized from competitor or intestine sample alone was used. The PCR cycle was as follows: initial denaturation for 3 min at 94°C, followed by 44 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 45 sec in a thermocycler (Robocycler). Starting at cycle 16 and at intervals of two cycles thereafter, 5% (v/v) portions of the reactions were removed and resolved on a 2% (w/v) ethidium bromide-agarose gel. Band intensities were quantified using Image Master (Amersham-Pharmacia) and plotting of optical density as a function of cycle number.

Analysis of sbPRLR in the Gonads by C-RT-PCR

Ten-fold serial dilutions in triplicate of the competitor cRNA were reverse transcribed together with 5 µg of total RNA from gonads (n = 4 per group) followed by 30 cycles of PCR as described above. The target RNA was quantified by densitometry plotting of the ratio of competitor to target band intensity as a function of the initial amounts of the competitor cRNA added (10-fold dilutions ranging from 0.003 to 3 pmol). A ratio of 1 (equivalent concentrations) was taken to indicate that equal amounts of competitor and target RNA were present and was used to determine the amount of target RNA in the reaction.

Statistics

All data are expressed as the mean ± SEM. Levels of E2, testosterone, and ketotestosterone are in nanograms per milliliter of plasma. Levels of mRNA for PRL in Northern blots of pituitary extracts are normalized with ß-actin and are expressed as arbitrary units. Data from the kinetics of amplification are expressed as optical density units. Data from the C-RT-PCR are expressed as picomoles per microgram of total RNA and were log10 transformed before analysis. The overall significance of any differences was determined by ANOVA. The level of significance was 5%.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Plasma E2 Levels and GSI

In adults implanted with E2, circulating E2 steroid levels at the end of the experimental period were twice as high as in controls (control, 0.47 ± 0.14 ng/ml; E2, 0.93 ± 0.17 ng/ml). No significant differences in the GSI (control, 0.38 ± 0.07; E2, 0.30 ± 0.03) were observed between the two groups of fish. The majority of the gonad corresponded to testicular tissue in both control and E2-treated fish and accounted for more than 90% of the total organ. In juveniles, circulating E2 levels in E2-treated fish increased significantly (control, 0.30 ±. 0.03 ng/ml; E2, 2.13 ± 0.39 ng/ml); no differences were observed in GSI (1.4 x 10-4 ± 0.4 x 10-4) between both groups. No mortality occurred during both experiments.

Expression of PRL and sbPRLR

Northern blot analysis of pituitary total RNA of adults demonstrated a single PRL mRNA transcript of 1.35 kilobases (kb). Treatment with E2 did not alter transcript size or number. However, it did significantly reduce (by 50%) pituitary PRL mRNA compared to the control group (Fig. 1). In contrast, in juveniles, E2 treatment caused a significant 1.5-fold increase in pituitary PRL mRNA (Fig. 1), and a clear difference was found between the levels of PRL mRNA in juvenile and adult controls, with the latter group having a higher expression.



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  		FIG. 1. Messenger RNA levels of seabream PRL in Northern blots of pituitary extracts 1 wk after implantation of E2 in adult and juvenile seabream. The blot was hybridized with the full-length sbPRL probe and exposed for 1 h. Loading differences of mRNA were normalized with ß-actin and expressed as arbitrary units (n = 8, mean ± SEM); An * indicates a statistically significant difference (P < 0.05, ANOVA)

In the adult gonads, four PRLR transcripts of 1.1, 1.3, 1.9, and 2.8 kb (Fig. 2) were present. No significant differences were observed between control and E2-treated fish regarding the number, size, and abundance of transcripts. The smallest transcript (1.1 kb) was the most abundant (control, 86.5%; E2, 85.7%; 100% is the sum of all the receptor transcripts detected), followed by the 1.3-kb transcript (control, 10.7%; E2, 8.9%), the 2.8-kb transcript (control, 1.8%; E2, 3.4%), and the 1.9-kb transcript (control, 1.0%; E2, 2.0%).



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  		FIG. 2. Representative pattern of Northern blot analysis of mRNA (poly [A]+, 10 µg) from gonads 1 wk after implantation of E2 in adult seabream. The blot was hybridized with a sbPRLR probe and exposed for 1 wk. The arrows indicate the sizes of the transcripts

A validation experiment for the C-RT-PCR in which intestine total RNA and approximately equal quantities of competitor cRNA were used yielded the two expected products of 249 and 369 bp (Fig. 3a), as confirmed by sequencing. The linear portion of the amplification curves had very similar slopes (Fig. 3b), indicating that the primers had similar efficiencies with the competitor cRNA and the sbPRLR target. The plot of log ratio of sbPRLR:competitor vs. competitor cRNA was linear (r2 = 0.99) (Fig. 3, c and d). In adults, C-RT-PCR revealed that control gonads expressed sbPRLR at a very low level (0.001 ± 0.0002 pmol/µg total RNA) (Fig. 4). However, sbPRLR expression was significantly increased (by 50-fold, 0.049 ± 0.0006 pmol/µg total RNA) by E2. In contrast, E2 treatment caused a 24-fold decrease in sbPRLR mRNA in juvenile gonads compared to control fish (Fig. 4). A clear difference in control values of the receptor was registered between adult and juvenile animals.



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  		FIG. 3. Kinetics of amplification of the sbPRLR target and competitor. a) An aliquot of 3 µg each of sbPRLR total RNA from intestine and cRNA from competitor were added to a PCR. Starting at cycle 16 and at intervals of two cycles thereafter, 5% portions of the reaction were removed and resolved on a 2% ethidium bromide-agarose gel. The upper band corresponds to the competitor. Products corresponding to target gene (sbPRLR) and competitor are indicated. Following gel electrophoresis, the bands intensity was determined. Data are plotted as units of optical density versus cycle number. 1, Positive control for target gene; 2, positive control for competitor; m, molecular weight marker (1 kb plus, Gibco BRL). b) C-RT-PCR of the sbPRLR in total RNA from gonads of seabream. c) Representative gel for sbPRLR. Electrophoresis was performed on a 2% agarose gel. Lanes 1–4: each have the same amount of gonad total RNA (5 µg) and 10-fold serial dilutions of competitor; lane 5: positive control sbPRLR; lane 6: positive control competitor; lane 7: negative control. Note that the competitor was loaded in decreasing concentrations from left to right on the gel. m, Molecular weight marker (1 kb plus, Gibco BRL). d) The graphic corresponds to the standard curve for the quantification of the amount of sbPRLR (r2 = 0.99)



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  		FIG. 4. Quantitative analysis of mRNA levels of sbPRLR by C-RT-PCR in gonads 1 wk after implantation of E2 in adult and juvenile seabream. Levels of mRNA are expressed as pmol/µg total RNA (n = 4, mean ± SEM). An * indicates a statistically significant difference (P < 0.05, ANOVA between control and E2-treated fish)

Immunohistochemistry of sbPRLR in Seabream Gonads

For immunohistochemistry, only gonads from control animals were analyzed. The mature gonads were composed principally of testicular tissue, with abundant spermatozoa in the sperm duct (Fig. 5a). The ovarian region of the gonads was filled with oogonia and perinucleolar oocytes (Fig. 5a). Seabream PRLR was detected in both testis and ovarian tissue (Fig. 5, b and c). Immunoreactivity in testis was most intense in the spermatogonia, particularly in the region surrounding the nucleus, although the heads of spermatozoa also stained less intensely. In the oocytes of the ovarian tissue, the staining pattern was cytoplasmic, and a strong reaction was found in the region nearest to the perinuclear membrane (Fig. 5c).



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  		FIG. 5. Immunohistochemical localization in gonadal sections from the seabream (Sparus aurata) PRLR. a) Section of ovotestis stained with hematoxylin and eosin. OVc, Ovarian cavity; Sd, sperm duct; SZ, spermatozoa; T, testis. b) Mature testis. A positive immunoreaction is present in spermatogonia (SG) and the heads of some spermatozoa (SZ). c) Ovarian tissue with a positive reaction in the oocytes. A strong signal is observed in some oocytes in the perinuclear region (Po). d) Control section in which normal swine serum replaced the primary antiserum was completely unstained. Magnification x20 (a) and x200 (b–d)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Despite the well-known roles of PRL in the reproductive physiology of higher vertebrates, most of the identified hormonal functions in fish have been in hydromineral balance (e.g., [12]). The present study provides strong evidence for a role of PRL in fish reproduction. Treatment with E2 resulted in a significant increase in pituitary PRL mRNA in juvenile gilthead seabream, and this agrees with a previously observed, dose-dependent increase in pituitary synthesis of two forms of PRL in E2-treated tilapia in vivo and in vitro [24, 25]. However, in rainbow trout (Oncorhynchus mykiss), E2 injection (5 µg/g body weight) did not result in a significant increase in either PRL mRNA levels or protein levels in the pituitary after 3 days of treatment in immature fish or after a 3-wk treatment period in mature male fish [26]. This was taken to suggest that PRL cells are not regulated by estrogens in this species [26]. The dosage and duration of the experiments may nevertheless explain the conflicting results.

The observed increase in PRL mRNA in juvenile gilthead seabream could be explained by either a direct action of E2 at the level of the pituitary cells and/or an indirect action via a hypothalamic route and GnRH neurons. A direct action on PRL cells is supported by the presence of estrogen-response elements in the upstream regulatory regions of the rat PRL gene [27–29], and mammalian lactotrophs and gonadotrophs have been shown to express estrogen receptor (ER) [30–33]. Whereas to our knowledge the PRL gene of gilthead seabream has yet to be characterized, {alpha} and ß ERs have been identified in the adult gilthead seabream pituitary [34] as well as in other teleosts (e.g., [35, 36]). Prolactin cells also have GnRH receptors [37], and GnRH has been shown to stimulate PRL release in several vertebrates, including fish [38]. Furthermore, E2 strongly increases GnRH fibers and pituitary innervation in immature African catfish (Clarias gariepinus), and an estrogen-response element has been found in the human GnRH gene [39]. However, GnRH neurons do not appear to express ER{alpha} [40]. In addition, a homologue of mammalian PRL-releasing peptide (PRP) has been isolated from tilapia brain, characterized, and shown to significantly increase circulating PRL levels [41]. Whether the neurons producing PRP could be a target for E2 needs investigation. In contrast to juvenile fish and mammals, in which E2 has a stimulatory effect on PRL gene transcription [19], in adult seabream it caused a drastic reduction in PRL gene expression. A range of factors may explain this difference, although it is not possible from the present results to determine the underlying cause(s). However, the differing hormonal status of the mature adult and immature juvenile seabream (e.g., E2 levels, present study; [21]) and differences in regulation of the pituitary gland during maturation of the gonads may in part contribute to the response observed. In fact, one of the characteristic features of juvenile vertebrates is the quiescent state of activity of the brain-pituitary-gonad axis as a functional unit [42, 43]. The difference in the status of the gonads may also be a contributing factor: adult seabream had fully mature gonads, several of which were actively spermiating, whereas the juvenile seabream had small, immature gonads, as shown by the GSI. Further studies will be required to elucidate the answers to this question.

The increase in PRL mRNA in juveniles does not necessarily indicate that PRL protein is also increased, but the decrease in gonadal sbPRLR mRNA measured in juveniles may be induced by negative feedback. In adult gonads, sbPRLR mRNA increased, possibly because of a compensatory mechanism for decreased pituitary expression. The difference between fish species may be explained again by the developmental status of the brain-pituitary-gonad axis, which in African catfish is more sensitive to positive feedback of sex steroids during puberty [42, 43]. In mammals, E2 stimulates the expression of PRLR in various tissues (e.g., [44, 45]), but differential regulation also increases expression in the mammary gland, but not in the liver, with the decline of progesterone at the end of pregnancy [46].

At the level of the gonads, several transcripts of seabream PRLR mRNA of 1.1, 1.3, 1.9, and 2.8 kb were detected. The presence of numerous PRLR transcripts is in direct contrast to previous observations in tilapia (Oreochromis niloticus, 3.3 kb [47]) and in rainbow trout (3.4 kb [48]), in which a single transcript was found in both male and female gonads. Surprisingly, in the goldfish (Carassius auratus), no transcripts were observed in either male or female gonads after Northern blot analysis using 5 µg of poly(A)+ mRNA [49]. The pattern of PRLR expression in seabream is more like that in other vertebrate groups, in which more than one PRLR transcript is present in the gonads (e.g., testis of ram, transcripts of approximately 3.6, 11.2, 12.6, and 14.2 kb [50]; testis of sexually mature chicken, transcripts of 1.2, 1.7, 2.0, 3.6, 4.6, and 11.7 kb [51]). Both in chicken and seabream, the 1.2-kb transcript was the most abundant. In addition, adult chickens produced three distinct, truncated, testis-specific cPRLR transcripts, of which two lacked regions coding for the extracellular and transmembrane domains [51]. The functional significance of this is unclear. In mammals, the PRLR gene consists of 11 exons [52, 53], which generate alternative splicing long and short forms of the receptor, which differ only in the intracellular domains [7]. The genomic organization of the PRLR gene in the seabream is not yet known, but the multiple transcripts observed in the gonads possibly may result from alternative splicing.

Even though the exact role of PRL in testicular/ovarian function is still controversial, the expression of PRLR protein in the gonads, as detected by immunohistochemistry, strongly supports the suggestion that PRL is acting directly on these tissues, possibly in spermatogonia, spermatozoa, and oocytes.

In summary, the present study shows that E2 affects PRL expression in the pituitary and PRLR gene expression in the pituitary and gonads, depending on the stage of maturity. Moreover, in the gonads, four different transcripts were identified, and the characteristics of these receptor transcripts are now under investigation. Finally, the localization of the receptor in both male and female gonadal tissue clearly supports the hypothesis that PRL is involved in reproduction in the seabream and may play a role in gametogenesis.


   ACKNOWLEDGMENTS
 
The authors thank Carlos Vale and Nádia da Silva (Universidade do Algarve) for carrying out Northern blot analysis and immunohistochemistry, respectively.


   FOOTNOTES
 
1 Supported by Fundação para a Ciência e Tecnologia, project Praxis/2/2.1/BIA/211/94. J.E.B.C. and C.R.A.S. are in receipt of grants SFRH/BPD/5555/2001 and Praxis/BPD/22040/99, respectively. Back

2 Correspondence: Deborah Power, Universidade do Algarve, CCMAR, FCMA, Campus de Gambelas, 8000-117 Faro, Portugal. FAX: 351 289 818353; dpower{at}ualg.pt Back

Received: 10 July 2002.

First decision: 31 July 2002.

Accepted: 22 August 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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