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Department of Aquaculture,3 National Taiwan Ocean University, Keelung 202, Taiwan
National Museum of Marine Biology and Aquarium,4 Pintung 944, Taiwan
Department of Aquaculture,5 National Kaohsiung Marine University, Kaohsiung 811, Taiwan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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luteinizing hormone, gonadotropin-releasing hormone, neuroendocrinology, pituitary, seasonal reproduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Black porgy, Acanthopagrus schlegeli Bleeker, a marine protandrous hermaphrodite, is widely distributed and is of particular interest in commercial aquaculture in parts of Asia [6]. Black porgy in Taiwan have an annual reproductive cycle with a multiple spawning pattern that occurs in late winter and early spring (December–March). Sex differentiation into the male sex occurs in 3- to 4-mo-old juvenile fish (unpublished data). Testicular tissue is the major gonadal tissue in juvenile fish to those aged 1 yr. Bisexual structures (containing both testicular and ovarian tissue) are characteristic of gonads from 1+- and 2+-yr-old black porgy. The fish are functional males for the first 2 yr of life but they begin to change sex during the third year. However, only about 40% of cultured black porgies change into females, whereas the rest remain as functional males during the third spawning season [7]. The mechanism of natural sex change in black porgy is important, but it is still far from clear. The seasonal concentrations and forms of GnRH in the brain and pituitary, and the profiles of plasma LH in black porgy and other perciform fish have been only briefly investigated [4, 5].
LH secretion in black porgy is reported to be regulated by an estrogen-specific effect [8, 9]. Seasonal changes of estradiol-17ß (E2), testosterone, and 11-ketotestosterone levels in plasma have been investigated in 1- to 3-yr-old black porgy [6, 7, 10–12]. High levels of plasma E2 during the prespawning and spawning seasons are correlated with the natural sex change in 3-yr-old black porgy [7]. E2 was found to induce sex change in 1-yr-old black porgy, however, the ovary remained at the primary oocyte stage [7, 10]. Vitellogenic oocytes were observed in 2-yr-old black porgy after treatment with E2 for at least 5 mo [11]. We therefore concluded that E2 may be important for natural and controlled sex change in protandrous black porgy [12].
Plasma LH levels ran parallel with E2-induced sex change in black porgy [13]. Short-term injection of E2 (but without changing sex status) also positively induced high levels of plasma LH [8, 9, 14]. The possible role of LH in the sex change of protandrous species is not yet clear. Exogenous hCG, LH, or GnRH have been reported to induce sex change in protogynous teleosts such as bluehead wrasse (Thalassoma bifasciatum) [15, 16] and ricefield eel (Monopterus albus) [17–19]. However, implantation of GnRH analog failed to induce sex change in 1+- and 2+-year-old protandrous black porgy [20, 21]. The possible role of the neuroendocrine system in the natural sex change process of protandrous fish (e.g., black porgy) and protogynous fish is a topic of great interest. But information on the changes that occur in neuroendocrine hormones in relation to the natural sex change is lacking in sex-changing fish, including black porgy. The bisexual gonad of the black porgy [6] is also an interesting model for the study of the endocrine mechanism of gonadal sex change. The hypothesis of this study was that the reproductive neuroendocrine system (especially the GnRH-gonadotropin axis) was involved in the natural sex change of protandrous black porgy.
To test this hypothesis, several studies were undertaken to compare the seasonal profiles of GnRH in the brain and pituitary of black porgy aged 2 mo to 2 yr; and seasonal plasma LH levels in juveniles (aged 2 mo to 1 yr) and those aged 1+ to 2 yr. The possible roles of LH were further analyzed by comparing plasma LH levels during the natural sex change period (from male to female) in fish aged 2+ to 3 yr. Reversible sex change (from an artificially sex reversed female to male) could occur naturally when E2-feeding was withdrawn before the spawning season [22]. Thus, the artificially sex-reversed female becomes a model fish for studying the mechanism of sex change. Therefore, the relationship between plasma LH levels and sex change (from artificially female to male) was further examined in the artificially sex-reversed female fish previously fed with E2. The possible roles of gonadotropin in the sex change were also investigated by examining the effects of hCG on gonadal aromatase activity and by the comparison of gonadotropin receptor transcripts between bisexual testicular and ovarian tissue.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fish used in the present study were aged 2 mo (mean body weight [BW] 1.57 ± 0.09 g in May), 1+ yr (mean BW 202.4 ± 7.3 g in August), and 2+ yr (mean BW 232.5 ± 15.4 g in March in the first experiment; 284.19 ± 13.67 g in May in the second experiment). Black porgy (A. schlegeli) were obtained from a pond culture. The fish were transported and acclimated to pond conditions at the university culture station (salinity 33 ppt; temperature 19–26°C). The fish were maintained in natural daylight and fed with commercial feed (Fwu Sou Feed Co., Taichung, Taiwan) at libitum. All procedures and investigations were reviewed and approved by the National Taiwan Ocean University Institutional Animal Care and Use Committee, and were performed in accordance with standard guiding principles for the care and use of laboratory animals.
Purification of GnRH in Brain and Pituitary
Brain and pituitary samples were collected from male fish aged 1+ yr (n = 6) during the prespawning season. The samples were homogenized in 1 N HCl, centrifuged, and neutralized. The extract was then purified with a C18 SPE column (Shandon Corp., Cheshire, England). The GnRH fraction was eluted with 30% acetonitrile in 0.12% trifluoroacetic acid (TFA; pH 6.5), dried, and dissolved with 0.12% TFA (pH 6.5). The GnRH extract was further purified by high-performance liquid chromatography (HPLC; Hitachi Corp., Tokyo, Japan) with a reverse-phase C18 column (250 x 4.6 mm i.d.; Kanto Chemical Corp, Tokyo, Japan). The elution gradients were, respectively, 0%–17% (0–10 min), 17%–24% (10–17 min), and then 24% acetonitrile in 0.12% TFA, with a flow rate of 1 ml/ min. Each fraction was dried and then dissolved in PBS pH 7.5 for GnRH radioimmunoassay (RIA). GnRH standards such as sbGnRH, mammalian GnRH (mGnRH), sGnRH, cGnRH-II, cGnRH-I, and lamprey lGnRH (lGnRH) were obtained from Bachem AG (Bubendorf, Switzerland).
Experimental Design
Four sets of experiments (I–IV) were conducted to study the seasonal changes in the profiles of GnRH in brain and plasma LH. The fish were grouped according to their age as juvenile (aged 2 mo to 1 yr), 1+ (aged 1 to 2 yr), or 2+ (aged 2 to 3 yr). The fish were anesthetized in 2-phenoxyethanol, and blood was taken with an EDTA-containing tube from the caudal vasculature. The plasma was separated by centrifugation and stored at –70°C for later LH analysis. The fish were killed by decapitation; brain and pituitary were quickly dissected from the juvenile and 1+ fish, and immediately stored with 1 N HCl at –70°C for subsequent GnRH RIA. Total body and gonadal weight were measured for the calculation of gonadosomatic index (GSI = gonadal weight/BW x 100%).
Seasonal Profiles of GnRH and LH in Fish Aged 2 Mo to 2 Yr (I)
The first experiment was conducted to study the seasonal change in the profiles of GnRH and LH in fish aged 2 mo to 2 yr. Twelve fish (in May, June, and July) and 8 fish (after July) were collected monthly from May (2 mo of age) to March of the next year; brain, pituitary, and blood samples were obtained for the GnRH RIA and LH RIA. In 1+ fish (n = 8), blood was collected monthly from September to May of the next year, whereas brain and pituitary samples were collected every 1.5 mo (September 4, October 17, November 28, January 9, February 20, March 31, and 15 May) for GnRH RIA. January–March is the spawning season for black porgy in the present study.
Plasma LH Profiles in Individually Marked and Naturally Sex-Changing in Fish Aged 2+ Yr (II)
To investigate the relationship between plasma LH levels and natural sex change, the plasma LH profiles during natural sex change were studied in 2+-yr males. Fish confirmed as males by the expression of sperm during the second spawning season were individually tagged, and blood samples were collected regularly at 2-wk intervals. In the first experiment, blood was collected from April to April of the next year (the end of the third spawning season) in fish aged 2+ to 3 yr; while in the second experiment, blood was collected from June (nonspawning season) to January of the next year (the third spawning season) in fish aged 2+ to 3 yr. The sexual status of each fish during the third spawning period was determined by biopsy (February in the first experiment, January in the second experiment), and accordingly, the fish were classified as either males or sex-changed females in the third spawning season. Thus, a total of 11 fish samples (6 from males and 5 sex-changed females) in the first experiment, and 15 fish samples (9 males and 6 sex-changed females) in the second experiment were obtained for the LH RIA.
Plasma LH Levels in Fish Previously Fed E2 (III)
Oral E2 induced an artificially sex-reversed female [11]. Reversible sex change (from an artificially sex-reversed female to male) could occur naturally when E2 was withdrawn before the spawning season [22]. Therefore, the artificially sex-reversed female could serve as a model fish for studying the mechanism of sex change. Two experiments (underyearling juvenile fish and those aged 1+ yr, respectively) were conducted by orally administering E2 (6 mg/kg of feed) to the fish ad libitum to induce sex change. E2 was fed for a period of 5 mo to fish aged 3 to 8 mo (from May to October) and to juveniles (underyearlings; n = 100), and for a period of 8 mo (from September to March of the next year) to the 1+-yr fish (n = 80). A control group of juvenile and 1+-yr fish were fed a diet devoid of E2. E2 feeding was withdrawn in juvenile fish during the early prespawning season (October) because the fish had changed to females by August based on biopsy and gonadal histology (i.e., they had become an artificially sex-reversed female) and they were later fed a control diet. In the 1+-yr fish, E2 feeding was withdrawn at the end of the spawning season (March) because the female gonad was observed from January (i.e., it became an artificially sex-reversed female). A standard period of 2 mo after sex change was set as the time for E2 withdrawal in a test group of both juvenile and 1+-yr fish. The sex status of the gonads in these fish was monitored for a period of 4–5 mo after E2 withdrawal by the expression of sperm and biopsy. This was also confirmed by histological observation [6] of the gonads at the termination of the experiment. Plasma (8 fish per group) was collected from juvenile fish in December, January, and March (after E2 withdrawal) for LH RIA, while the 1+-yr fish (8 fish per group) were bled in April, May, and July (after E2 withdrawal) for LH RIA.
Weekly Injection with hCG to Fish Aged 2+ Yr During the Nonspawning Season (IV)
The fourth experiment was conducted to investigate the possible role of gonadotropin in the natural sex change process in black porgy. It was performed by injecting the fish with hCG and monitoring gonadal aromatase activity. Gonadal aromatase activity is considered an early indicator for evaluating gonadal status and sex change [12, 13]. The test fish (2+ yr of age, n = 10; mean BW 260.5 ± 10.4 g) were injected with hCG (1 IU/g BW; Sigma, St. Louis, MO) at weekly intervals from May to the end of July (during the nonspawning season). Fish (n = 10) of the same age injected with saline served as controls. Gonadal tissue was collected at the end of treatment, and ovarian aromatase activity was measured using a radiometric method (1ß–3H androstenedione as the precursor) according to a previous study [13]. Aromatase activity was expressed as fmol 3H2O/ h·mg protein.
Analysis of GnRH and LH Levels Using RIA
Brain was homogenized and neutralized with NaOH in phosphate buffer. Specific RIAs for sbGnRH, sGnRH, and cGnRH-II were conducted. The sbGnRH antiserum was induced from a rabbit (by s.c. injection at multiple sites) against sbGnRH-hemocyanin of keyhole limpets (50 µg per injection) [23]. Specific antiserum against respective sGnRH and cGnRH-II was donated by Dr. K. Aida (University of Tokyo, Japan) and Dr. K. Okuzawa (National Research Institute of Aquaculture, Tamaki, Mie, Japan). Seabream GnRH, sGnRH, and cGnRH-II were iodinated with chloramine T and separated with a Sephadex G-25 column. The respective GnRH was used as a standard and 125I-GnRH as a labeled peptide in the RIA. Parallel binding was found in the respective sbGnRH, sGnRH, and cGnRH RIA in the reaction with different dilution of the extract of brain and pituitary in black porgy. All the samples were analyzed in one assay for GnRH RIA. The intraassay variability of sbGnRH, sGnRH, and cGnRH-II was below 10%. The cross-reactivity with sbGnRH, sGnRH, cGnRH-II, cGnRH-I, mGnRH, and lGnRH was as follows: 0.5% cGnRH-II and 0.05% lGnRH with sGnRH antiserum (sGnRH as 100%, no cross-reactivity with other GnRHs), 0.8% sGnRH with cGnRH-II antiserum (cGnRH-II as 100%, no cross-reactivity with other GnRHs), and no cross-reactivity of any other GnRH with sbGnRH antiserum (sbGnRH as 100%).
LH levels were measured by a homologous RIA (purified black porgy LH as a standard and anti-black porgy LH ß serum as antibody) [9]. Purified LH (5 µg) was iodinated by an iodogen-coated tube (Pierce, Rockford, IL). Free iodine was separated from the labeled hormone by gel filtration on a Sephadex G-50 column. All the samples in the same experimental group were examined in one assay for LH RIA, and intraassay variability was 10.2%. The cross-reactivity of black porgy FSH to LH RIA was below 1%.
Cloning of FSH Receptor and LH Receptor
Total RNA from testis was extracted by homogenization in Trizol reagent (Gibco-BRL; Grand Island, NY). Reverse transcription was performed using Superscript II (Gibco-BRL) with oligo (dT)12–18 primer under the following conditions: 42°C for 50 min, 37°C for 15 min, and 70°C for 15 min. The resulting cDNA served as template for subsequent polymerase chain reaction (PCR) amplification of gonadotropin receptors (FSH receptor and LH receptor) using two degenerated primers according to a previous study [24]: sense, 5'-GCNGANABRGCRSARAANGARATVGGNGCCATRCA-3'; antisense, 5'-GCNGAYGMBTTYAAYCCBTGYGARG-3'. The PCR reaction was performed in a final volume of 25 µl containing 2.5 µl of 10x reaction buffer, 1 µl of 10 mM deoxynucleotide triphosphate, 1 µl of 2 mM MgCl2, 30 pmol of each primers, and 1 U of Taq polymerase (Promega, Madison, WI).The PCR reaction was carried out for 35 cycles as follows: 94°C for 1 min, 50°C for 1 min, and 72°C for 4 min. The resulting PCR product of expected size (approximately 730 base pairs [bp]) was excised and purified using a Geneclean II kit (Bio 101, La Jolla, CA). PCR products were cloned into pGEM-T vector (Promega) and transformed in Escherichia coli competent cells following the manufacturer's instruction. White colonies were selected from X-Gal/IPTG ampicillin agar plates and grown in LB/ampicillin liquid media. Plasmid containing an insert was sequenced using a dye terminator cycle sequencing kit (Perkin Elmer, Foster city, CA).
LH Receptor and FSH Receptor Transcripts in Bisexual Gonad
Twelve black porgy (age 2+ yr; BW 352 ± 15 g, GSI 0.31 ± 0.04; n = 12) were collected in June–August (nonspawning season) to determine gonadotropin receptors. One microgram of total RNA from bisexual testicular and ovarian tissue was reversed-transcribed to the first-stranded cDNA using Superscript II with oligo (dT)12–18 primer. A PCR reaction was set with these cDNAs and the respective specific primers for FSH receptor and LH receptor. No PCR product was found in these RNA samples by adding the respective specific LH receptor and FSH receptor primers, indicating that the RNA extract was not contaminated with genomic DNA. A semiquantitative reverse transcription-PCR analysis from five representative fish was performed with specific primers designed for FSH receptor (nt 73–89, 5'-ATCCTCGCCCTGCTGGGG-3' and nt 564–585, 5'-ACAGAAGAAGGCCAGGATGTTG-3'), LH receptor (nt 368–385, 5'-CCAACGCCATGCACGTTA-3' and nt 657–709, 5'-CCATACACACAAAGTCGGTGAAA-3'), and ß-actin (nt 46–67, 5'-CTACAACGAGCTGAGAGTTGC-3'; nt 414–434, 5'-CACGTAGGAGAGCTTCTCCTT-3') in the following conditions: 30 cycles at 94°C for 30 sec, 50°C for 1 min, and 72°C for 1 min. This number of PCR cycles was preliminarily tested, and it was in the range of the linear curve in the relationship between the number of cycles and PCR product. About 513, 342, and 388 bp were specifically amplified in FSH receptor, LH receptor, and ß-actin, respectively.
Absolute quantification with real-time PCR analysis for gonadotropin receptors in fish aged 2+ yr (n = 12) was conducted according to the methods described in a previous study [25]. Specific primers were designed for the real-time PCR of FSH receptor (nt 342–359, 5'-CACCCTGGAGCGCTGGTA-3'; nt 389–409, 5'-CGTGTCTCAGGCGAAGTTTG-3') and LH receptor (nt 643–663, 5'-CGTCACGGAGACACCAAGATC-3'; nt 687–709, 5'-CCATACACACAAAGTCGGTGAAA-3'). Gene quantification of standards, samples, and controls was simultaneously conducted by real-time PCR (GeneAmp 5700 Sequence Detection System; Applied Biosystems, Foster City, CA) with SYBR green I as a double-stranded DNA minor-groove binding dye. The respective standard curve of log (transcript concentrations) vs. CT (the calculated fractional cycle number at which the PCR-fluorescence product is detectable above a threshold) was obtained. The correlation of the standard curve was –0.999. The values detected from different amounts of RNA (1 µg, 0.1 µg, 0.01 µg, and 0.001 µg) from the representative samples were parallel with the respective standard curve. No cross-reactivity was found between FSH receptor and LH receptor for the respective primers in semiquantitative PCR and real-time PCR.
Data Analysis
All data are expressed as the mean ± SEM. The difference in the profiles of GnRH and LH was subjected to a Duncan multiple range test after one-way analysis of variance (ANOVA) (P < 0.05) [26]. Data from tagged fish (see Fig. 6) were analyzed by repeated measures ANOVA. Logarithmic transformation was applied to the data before statistical analysis. A Student t-test was applied to compare the means of two samples.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GnRH in brain (sbGnRH, sGnRH, and cGnRH-II) and pituitary (sbGnRH and sGnRH) was detected using HPLC and RIA techniques (Figs. 1 and 2). Similar levels of sbGnRH and sGnRH but lower levels of cGnRH-II were found in brain (Fig. 1). Higher levels of sbGnRH were detected in the pituitary compared with those of sGnRH, but cGnRH-II was not detected in pituitary (Fig. 2).
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Seasonal Changes of Brain sbGnRH, sGnRH, and cGnRH-II
In juvenile fish, brain sbGnRH, sGnRH, and cGnRH-II concentrations increased in May, reaching high levels in July (cGnRH-II) or August (sbGnRH and sGnRH) (Fig. 3A). All three forms of GnRH decreased to their lowest levels in December and gradually increased from January to March (the spawning period) (Fig. 3A).
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In 1+-yr fish, high levels of brain sbGnRH were observed in September, November, and March (the spawning period), whereas high levels of sGnRH were observed in September, November, and February (the spawning period) (Fig. 3B). Brain cGnRH-II levels were consistently maintained at a low range from September to May (Fig. 3B).
Seasonal Changes of Pituitary sbGnRH and sGnRH
Very low levels of pituitary sGnRH were detected in juvenile fish. Pituitary sbGnRH levels gradually increased from July to October, reaching a plateau from October through March (Fig. 4A). Pituitary sbGnRH content was highest in September and March through May but low in January–February in 1+-yr fish (Fig. 5A). Low and relatively constant levels of sGnRH were detected in 1+-yr black porgy from September to May (Fig. 5A).
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Plasma LH Levels in Juvenile, 1+-Yr, and 2+-Yr Fish
Plasma LH levels were higher in January than in other periods in juvenile fish, when GSI was the highest in December–January (Fig. 4B). In 1+-yr fish, plasma LH levels were higher in September–October and March–April compared with other sampling times (Fig. 5B).
Plasma LH levels increased during the prespawning and spawning periods (December–January) in both 2+-yr males and females in which samples were collected from April through the following April (over a 1-yr period) and from June to January (over a 7-mo period) (Fig. 6, A and B). Significantly lower levels of plasma LH were detected in sex-changing fish (male to female) during the nonspawning period (July and September in experiment I, and July in experiment II) compared with the fish that remained in the male phase in the first and second experiments (Fig. 6, A and B).
Plasma LH Levels Associated With Sex From an Artificially Sex-Reversed Female to Male
E2 induced an artificially sex-reversed female in juvenile and 1+-yr fish. E2 feeding was withdrawn during the early prespawning season (October) in juvenile fish. After 2 mo of E2 withdrawal, testicular tissue with sperm was found in the gonads of previous E2-fed juvenile fish in December, and the artificially sex-reversed female became male in December–January. Plasma LH levels were higher in the previous E2-treated group than in the control group in December and January (after 2–3 mo of withdrawing E2 administration) (Fig. 7A).
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E2 feeding was withdrawn at the end of the spawning season (March) in 1+-yr fish. In this previous E2-fed group, gonadal sex remained in the female phase for 4 mo (March–July) after E2 withdrawal. No difference in plasma LH levels was found in the control and previous E2-treated group in April, May, and July (Fig. 7B).
Gonadal Aromatase Activity in hCG-Treated Fish
No gonadal sex change was observed at the end of July in fish receiving weekly injections of hCG for 10 wk. There was no gross difference in the gonad after biopsy (in relation to the ratio of ovarian and testicular tissue) in hCG and control groups. The increase of the ovarian portion in the bisexual gonad during the nonspawning season made it the major tissue in the gonad in comparison to testicular tissue. Therefore, aromatase activity was measured in the gonad instead of just ovarian tissue. GSI between the control (0.46% ± 0.06%) and hCG-treated (0.34% ± 0.08%) groups was not significantly different. Similarly, gonadal aromatase activity was also not significantly different between the control (86.9 ± 11.9 fmol 3H2O/h·mg protein) and hCG-treated (73.9 ± 6.6 fmol 3H2O/h·mg protein) groups.
Differential Expression of FSHR Receptor and LH Receptor in Bisexual Testicular and Ovarian Tissues During the Nonspawning Season
Partial cDNA of FSH receptor (GenBank accession number AY598753) and LH receptor (GenBank accession number AY596169) was cloned from the gonad of black porgy. The homology comparison of the deduced amino acid sequence was 57%–74% in FSH receptor and 56%– 83% in LH receptor with other teleosts such as tilapia (GenBank accession numbers AB041762 and AB041763), amago salmon (GenBank accession numbers AB030012 and AB03005), African catfish (GenBank accession numbers AJ012647 and AF324540), and zebrafish (GenBank accession numbers AY424301 and AY424302).
FSH receptor and LH receptor were differentially expressed in testicular and ovarian tissue of the bisexual gonad in fish aged 2+ yr (cDNA was pooled from five fish) during the nonspawning season according to semiquantitative PCR results (Fig. 8). Real-time PCR analysis showed higher expression levels of FSH receptor transcripts (27-fold) and LH receptor transcripts (19-fold) in bisexual testicular tissues compared with those in the respective ovarian tissues (n = 12 fish) (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This is the first report that shows long-term seasonal changes in GnRH levels in the brain and pituitary, and LH in plasma in black porgy. All three forms of GnRH were detected in the brain of 2-mo-old fish (mean BW 1.57 g) with increased levels from May to August. In gonochoristic fish, the involvement of GnRH in promoting sex differentiation was suggested in tilapia [27, 28] and sea bass (Dicentrarchus labrax) [29]. The highest GnRH levels were detected in the pituitary of European sea bass (D. labrax) when the gonads started to differentiate [29]. In black porgy, the gonadal sex differentiation occurs from June to August (3 mo to 5 mo of age; unpublished data) and thus the increase in brain GnRH could be related to the occurrence of sex differentiation in juvenile black porgy.
Fluctuating concentrations of brain sbGnRH and sGnRH were observed in black porgy aged 1+ yr, with high levels of brain sbGnRH during the nonspawning (September), prespawning (November), and spawning (March) seasons. In red seabream (P. major), telencephalon, brain, and pituitary sbGnRH levels were reported to reach a peak in April (spawning season) [4, 5]. In goldfish, there were no clear parallel changes in brain GnRH levels with seasonal ovarian development [30]. In masu salmon (Oncorhynchus masou), the brain sGnRH contents increased during the underyearling phase and fluctuated thereafter, but pituitary sGnRH content increased in accordance with maturation [31]. In the present study, pituitary sbGnRH content in black porgy increased during growth in underyearling black porgy and reached a plateau in January. Pituitary sbGnRH content was closely associated with plasma LH levels in underyearling and 1+-yr black porgy. The data support the concept that sbGnRH is the physiological and main form of GnRH regulating gonadotropin concentrations in black porgy. Seabream GnRH has also been indicated as the most important hypophysiotropic form of GnRH in gilthead seabream (S. aurata) [2], red seabream (P. major) [4], and European sea bass (D. labrax) [29]. A positive correlation between pituitary sbGnRH levels and plasma LH levels was also found in gilthead seabream (S. aurata) [32].
A high content of pituitary sbGnRH was observed during the nonspawning and spawning seasons in black porgy aged 1+ yr. The significance of the increased pituitary sbGnRH content and plasma LH levels during the nonspawning season in fish aged 1+ yr remain unclear. Black porgy is a protandrous hermaphrodite; all the fish reach puberty and become functional males during the first and second spawning seasons. For the 1+-yr black porgy in the present study, development of the male phase in the second spawning season was associated with elevated levels of plasma LH during the previous nonspawning season. Two separated experiments were further conducted for fish aged 2+ yr to investigate the association of plasma LH profile and sex status. Similar profiles of plasma LH concentrations were consistently found in these two experiments (i.e., a high plasma LH concentration during the nonspawning period if the fish remained in male phase during the third spawning season). The higher levels of plasma LH in these males aged 2+ yr (compared with the sex-changing females) were comparable to those of increased plasma LH and sbGnRH content in brain and pituitary of those aged 1+ yr during the nonspawning season. Thus, the data reveal that elevated levels of plasma LH and pituitary sbGnRH (during the nonspawning season) were consistently associated with the development of testicular tissue and the male phase in the coming second or third spawning season.
Data from the artificial sex reversal study provided further evidence for the association of high plasma LH levels with the development of the male phase. Effects of E2 on induced sex change (artificially sex-reversed female) in the present study was consistent with our previous studies [10, 11]. However, we also found that E2 could induce a reversible sex change if E2 feeding and sex change were completed during the prespawning season [22]. This interesting phenomenon (reversible sex change) in black porgy will provide a unique model for further studying the association of plasma LH levels with the process of sex change (from female to male). In the experiment with juvenile fish, we found high levels of plasma LH in the naturally sex-changing fish from an artificially sex-reversed female to male (Fig. 7A). In contrast, no significant difference in plasma LH levels was observed in the previous E2-fed fish compared with those of control 2-yr-old fish (Fig. 7B). No natural sex change (from an artificially sex-reversed female to male) was found in these previous E2-treated fish because the period of E2 feeding lasted until the end of the spawning season. A parallel relationship among high gonadal aromatase activity, elevated plasma LH levels, and induced sex change was found in E2-fed (aged 1+ yr) fish in our previous studies [12, 13]. We also found that short-term injection with E2 (but without sex change) caused increased plasma LH levels [8, 9, 14]. Therefore, the elevated plasma LH level [8, 9, 12–14] may be related to the positive control of E2 action on LH, and it may not be directly associated with the inducement of sex change (from male to female). In contrast, according to our present data, plasma LH levels were related to male development.
Higher levels of plasma E2 and vitellogenin were found simultaneously in the naturally sex-changing female black porgy compared with that of male fish (2+ to 3 yr of age) [7]. Significantly elevated plasma testosterone levels but with no change in plasma E2 were detected in the artificially sex-reversed female (1+ yr of age) after E2 feeding was withdrawn. Furthermore, reversible sex change (from artificial female to male) also was found to occur in these fish as a result of E2 withdrawal [22]. The profiles of increased plasma testosterone were consistent with the natural sex change from artificial female to male [22]. However, in the present experiments, plasma sex steroids could not be measured in addition to plasma LH because of inadequate plasma sample volume.
Bisexual gonads (with ovarian and testicular tissue together) were found in the black porgy before sex change [6]. Ovarian tissue became dominant in fish from the postspawning to the nonspawning season in 1+- and 2+-yr black porgy; in contrast, ovarian tissue regressed and testicular tissue developed to become dominant in fish from the nonspawning to the spawning season in 2- and 3-yr-old black porgy [6]. Histological data revealed the process of a gradual development of bisexual gonad to the primary oocyte-gonad stage during natural sex change in 2+-yr-old black porgy. This is accompanied by the complete regression of testicular tissue in October (early prespawning season) and further development of ovaries to the vitellogenic stage in the approaching spawning season (December– March) [33]. Thus, it is suggested that the increase of plasma LH levels during the nonspawning season (June–August) maybe related to the development of testicular tissue in the bisexual gonad. In contrast, low and constant levels (basal levels) of plasma LH during the nonspawning season cause testis regression, which in turn, possibly removes a testis-induced inhibition on ovary growth, leading to ovarian development. The testicular tissue is considered to be more sensitive than ovarian tissue to stimulation by gonadotropins. FSH, LH, or both probably stimulated testicular tissue growth, but not that of ovarian tissue. Different levels of gonadotropin receptors may account for the differential sensitivity in testicular and ovarian tissues of the bisexual gonad. This study further demonstrates that LH receptor and FSH receptor transcripts were possibly more expressed in the testis than in the ovary of the bisexual gonad of 2+-yr-old fish. Earlier studies conducted on black porgy have shown that bisexual testicular tissue has higher levels of androgen receptors and estrogen receptors than does ovarian tissue [25]. Our present investigation yields the first finding on the differential responsive activity between testicular and ovarian tissue based on the expression of gonadotropin receptors and other hormone receptors.
The number of preoptic GnRH neuron cells was also significantly higher in males than in females of protogynous bluehead wrasse (T. bifasciatum) [34], ballan wrasse (Latrus berggylta) [35], and protandrous dusky anemonefish (Amphiprion melanopus) [36]. Exogenous hCG, LH, or GnRH induced gonadal sex change in two protogynous species, bluehead wrasse, T. bifasciatum [15, 16] and ricefield eel, M. albus [17–19], but not in protandrous black porgy [20, 21]. Therefore, it seems that the neuroendocrine system (GnRH-gonadotropin) is involved in the controlled sex change in the male phase in protogynous hermaphroditic fish. To the best of our knowledge, this study is the first report demonstrating the correlation between natural sex change and the profiles of plasma LH in a hermaphroditic species. To better understand the endocrine mechanism in both natural and artificial sex change in black porgy, further studies are needed to provide direct evidence for the association of plasma LH and the male phase. Currently, the possible role of FSH in the sex change process is not known because an FSH RIA is not available for this species.
Gonadal aromatase activity has been shown to have a relationship with ovarian development in several vertebrates. Gonadal aromatase activity was higher in vitellogenic fish than in mature salmon (O. rhodurus) [37]. Masculinization of the gonad by aromatase inhibitors has been shown in gonochoristic chinook salmon (O. tshawysha) [38] and lizards [39]. Gonadal aromatase activity had been indicated to play an important role in the sex differentiation and female development in chickens [40], tadpoles [41], and teleosts [42]. Gonadal aromatase activity has also been reported to associate with the induced sex change in black porgy [12, 13]. E2 administration for 1.5 mo to induce sex change also significantly stimulated gonadal aromatase activity in black porgy [13, 43]. Gonadotropin stimulates aromatase activity in the ovary of gonochoristic fish. Ovarian aromatase activity was stimulated by the injection of eCG in medaka, Oryzias latipes [44]. LH stimulated E2 production (an increase in aromatase activity) in medaka [44] and red seabream [45, 46], and even LH had a much higher potency than FSH in red seabream [46]. Therefore, gonadal aromatase was considered as an early bio-indicator for evaluating the effects of exogenous gonadotropin in sex change in this study. Long-term (10 wk) treatment with hCG during the nonspawning season failed to induce higher gonadal aromatase activity and ovarian development. The data suggest that exogenous gonadotropin given during the nonspawning season probably fail to induce sex change in black porgy. The lack of increase in ovarian aromatase activity by the exogenous gonadotropin is also supported by the previous studies in which hCG stimulated the increase in plasma testosterone levels but not E2 during the nonspawning season in bisexual 2+-yr-old black porgy [47]. In contrast, hCG stimulated the increase in plasma testosterone and E2 levels in females during the spawning season [47]. But in the current experiment, hCG treatment did not induce a growth of testicular tissue in the bisexual gonad. Further studies (related to the dosage, timing, and duration of treatment) are required to understand the roles of exogenous gonadotropin in the development of bisexual ovary/ testis.
In conclusion, the examination of seasonal profiles of GnRH concentrations from juvenile age to 2 yr in black porgy showed that the increase in brain GnRH was correlated with gonadal sex differentiation. Brain sbGnRH, sGnRH, and cGnRH-II levels were high during the nonspawning and spawning seasons. The levels of pituitary sbGnRH and plasma LH were found to be high during the nonspawning and spawning seasons. The levels of plasma LH were higher during the nonspawning season in 2+-yr-old males than in sex-changing females. Therefore, high and basal levels of LH during the nonspawning season are suggested to favor the development of male and female gonad, respectively. That high levels of plasma LH favored the development of the male phase was confirmed in the present experiments as follows: the natural sex change from an artificially sex-reversed female to male in relation to plasma LH levels, higher FSH receptor and LH receptor transcripts in the bisexual testicular tissue compared with that of ovarian tissue, and the failure to stimulate the increased activity of gonadal aromatase by hCG. Seasonal profiles of GnRH-LH will provide a better understanding of the mechanism of sex change in black porgy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. Fax: 886 2 2462 1579; b0044{at}mail.ntou.edu.tw
Received: 15 June 2004.
First decision: 14 July 2004.
Accepted: 14 December 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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anós E, Holland MCH, Zohar Y, Zanuy S. Pituitary levels of three forms of GnRH in the male European sea bass (Dicentrarchus labrax, L.) during sex differentiation and first spawning season. Gen Comp Endocrinol 2000 120:67-74[CrossRef][Medline]
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