| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a National Museum of Marine Biology and Aquarium, Taiwan, Republic of China
b Department of Aquaculture, National Institute of Kaohsiung Marine Technology, Taiwan, Republic of China
c Department of Aquaculture, National Taiwan Ocean University, Taiwan, Republic of China
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
aromatase, luteinizing hormone, male sexual function, steroid hormones
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Plasma estradiol-17ß (E2), LH (gonadotropin II), and gonadal aromatase were associated with the controlled sex change in protandrous black porgy according to our previous studies [2, 4–8]. The mechanism of natural sex change in black porgy is important but it is still far from clear. According to previous data on the controlled sex change of black porgy, we further propose that aromatase plays a key role in the natural sex change in this protandrous fish.
The major role of estrogens and aromatase in female sex differentiation has been studied by aromatase inhibitor (AI) treatment in other vertebrates. A 2-h treatment after hatching with the nonsteroidal inhibitor fadrozole caused the completion of sex reversal in female embryos in a gonochoristic fish, chinook salmon Oncorhynchus tshawaytsha [9]. In the amphibian Rana catesbeiana, steroidal AI 4-hydroxyandrostenedione induced transformation of ovaries into testes [10]. In Alligator mississipiensis, fadrozole had a weak effect on ovarian development [11]. Treatment with nonsteroidal AIs (fadrozole or letrozole) also induced masculinization in turtles [12–14], birds [15], and newts Pleurodeles waltl [16]. Not many data are available to demonstrate the functional role of aromatase in the natural sex change of sex-differentiated animals. The possible mechanism of the natural sex change is still unclear in hermaphrodite species.
Therefore, the objectives of this study were to investigate the control of sex status by a long-term treatment with AIs in 2+-yr-old protandrous black porgy. The effects of AIs in male function (plasma 11-ketotestosterone [11-KT] and milt volume) and LH were also studied.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Two-year-old functional male black porgy, A. schlegeli (mean body weight, 296.2 ± 11.8 g) were obtained from a pond culture in February 1999. Milt was obtained by hand stripping each functional male at the beginning of the selection process of the experiment. All experimental fish were acclimatized to a pond at a university culture station in seawater and a natural light system. The water temperature ranged from 20°C to 27°C. The fish were fed semidried pellet feed prepared from commercial powder feed (feed composition: protein, 44%; lipid, 3%; cellulose, 1.2%; and ash, 16.5%; Fwu Sou Feed Co., Taichung, Taiwan). The fish were handled in an appropriate way to fulfill animal experimentation procedures.
Oral Administration with Aromatase Inhibitors
In order to investigate the effects of aromatase inhibitors in sex change and milt volume, functional male fish (n = 70) were divided into two groups, a control group (n = 35) and an AI group (fadrozole, 4-(5,6,7,8-tetrahydroimdazole [1,5-a]pyridin-5-yl)-benzonitrile monohydrochloride and ATD, 1,4,6-androstatriene-3,17-dione; Steraloids Inc., Wilton, NH). Each group had two tanks (2.5 tons of water in a fiberglass tank, 17–18 fish per tank) in a seawater system. Each tank had its own recirculating system. Fadrozole and ATD were prepared in a stock solution (10 mg/ml) with distilled water and absolute alcohol, respectively. Each AI (10 mg fadrozole/ml + 10 mg ATD/ml) was mixed with 1 kg of powder feed and water, then the feed was made into pellets and dried. Fadrozole was donated by Novartis Pharmaceuticals Co., Summit, NJ. AIs were administered ad libitum from April 1999 to January 2000.
In the experiment of aromatase effects on sex change, fish in each group were collected monthly, bled, and tested for spermiation-milt volume. The sampling time occurred at 1500–1700 h to prevent the possible changes due to the effects of diurnal rhythms. At the end of the experiment, 14 fish in the control and 21 fish in the AI-fed groups remained alive and were used in the experiments. Fish were anesthetized, bled, and killed by decapitation. Gonadal status was also determined at the end of the experiment. The average amount of ingested aromatase inhibitors in each fish was estimated as 15.2 mg during the experimental period (from March 1999 to January 2000).
Sampling Procedures
The fish were anesthetized in 2-phenoxyethanol during bleeding, milt collection, and killing. Blood (n = 14 from each group) was taken with an ethylenediamine tetraacetic acid (EDTA)-containing tube from the caudal vasculature. Spermiation in fish was defined as milt that could be collected from the genital pore by hand stripping, and milt volume (n = 12 per group) was measured with a syringe. The plasma was separated by centrifugation and stored at -70°C for later analysis of LH and 11-KT. The gonad, pituitary, and brain were quickly dissected and weighed to measure aromatase activity at the end of experiment. Total body and gonadal weights were measured for calculating the gonadosomatic index (GSI = gonadal weight/body weight x 100%).
Measurement of Aromatase Activity
Aromatase activity (5 females and 9 males in the control group; 8 males in the AI group) in the gonad, pituitary, and brain (forebrain, midbrain, and hindbrain) was measured using a radiometric method modified from previous studies [7, 17]. The stereo-specific loss of hydrogen from the C-1ß position of 1ß-3H androstenedione (3H-A) during aromatization and the formation of H2O was measured. Gonads and brain were homogenized with a potassium phosphate buffer and centrifuged at 1000 x g for 10 min at 4°C. The crude supernatant fraction was added to 100 µl of cofactor solution and 0.3 µM 3H-A (555 GBq-1.11 TBq; Dupont Co., NEN Research Products, Boston, MA). The reaction solution was incubated at 25°C for 80 min and stopped by addition of 10% trichloroacetic acid. After centrifugation, the supernatant solution was twice subjected to charcoal extraction (80 mg charcoal/ml in 10% trichloroacetic acid) to remove the excess unreacted 3H-A. The radioactivity of each fraction was measured using a liquid scintillation counter (Wallac 1409, Pharmacia, Finland). The assay was conducted in duplicate. Aromatase activity was expressed as pmol 3H2O/h mg protein.
LH and 11-KT Assay
Plasma LH (n = 14 in both AI and control groups) was measured with a homologous radioimmunoassay (RIA) as described in previous studies [18]. Plasma 11-KT (n = 9 in both AI and control groups) was measured by RIA following diethyl ether extraction as previously described [5]. The samples were analyzed within the same batch of assay in duplicate tubes. LH and 11-KT antisera had a cross-reactivity of less than 1% and 5% against FSH and testosterone, respectively. The sensitivity of the LH and 11-KT RIAs was 0.25 ng and 5 pg, respectively; the intraassay variation in both assays was less than 12%.
Data Analysis
Two-way ANOVA was conducted to test significant interaction between treatment (AI) effect and timing (season). One-way ANOVA followed by a Student-Newman-Keuls multiple comparison test examined the significance between treatment at a single time and in the effect of specific treatment through time (P < 0.05) [19]. Logarithmic transformation was applied to the data (Figs. 1–3) before statistical analysis. Results are expressed as mean ± SEM.
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All the AI-fed fish (n = 21, 100%) remained as functional males with active spermiation during the spawning season. These male fish showed spermiation as early as October 1. In the control groups (n = 14), fish were females (n = 5; 35.7%), and functional males (n = 9; 64.3%). The spermiation in the control males started in early January, about 3 mo later than the AI males. The average amount of milt volume in a control male was significantly less than for AI males (Fig. 1). AI treatment and season had a significant interaction in milt volume (F(7, 176) = 30.89, P < 0.01). The GSI in the AI-males, control males, and control females was 2.44% ± 0.37%, 0.72% ± 0.11%, and 1.91% ± 0.48%, respectively. AI-males had a significantly higher GSI than the control males (P < 0.05).
11-KT and LH Concentrations
After 3 mo of treatment, plasma 11-KT levels were significantly higher in the AI group than in controls. Three high peak levels of 11-KT were detected in October, November, and December, respectively, in the AI-treated group (Fig. 2). Higher plasma 11-KT levels in the AI-fed group than in the control group were detectable during the nonspawning season (June–September; Fig. 2). AI treatment and season had a significant interaction in plasma 11-KT (F(14, 240) = 8.31, P < 0.01). Plasma LH concentrations were higher in AI fish than in controls in May, and August–November (Fig. 3). AI treatment and season had a significant interaction in plasma LH (F(15, 416) = 7.52, P < 0.01). No difference (P > 0.05) in pituitary LH content was found in AI-treatment or control groups (5.00 ± 0.47 µg/pituitary in the AI fish, 5.29 ± 0.42 µg/pituitary in the control fish).
|
|
Aromatase Activity in the Brain, Pituitary, and Gonad
No difference (P > 0.05) in aromatase activity (pmol/h · mg protein) in the forebrain (54.9 ± 7.8 vs. 50.2 ± 3.1), midbrain (11.2 ± 1.9 vs. 9.9 ± 0.6), hindbrain (1.8 ± 1.3 vs. 0.5 ± 0.2), and pituitary (24.3 ± 3.3 vs. 23.3 ± 3.8) was detected in the females and males, respectively, of the control group. Therefore, data from control females and males were pooled as the control (Fig. 4). Aromatase activity in forebrain, midbrain, and hindbrain was suppressed in the AI fish compared with the control group (Fig. 4). The degree of suppression was 5-fold in forebrain, 5.2-fold in midbrain, and 7.2-fold in the hindbrain, respectively (Fig. 4). Feeding with AIs also had a 3-fold suppression in the pituitary aromatase activity compared with controls (Fig. 4). In the control group in January, female gonad (ovary) had a higher (P < 0.05) aromatase activity than male testis (Fig. 5). The male gonad (functional testis) in the AI-fed group had the lowest aromatase activity compared with control males and females at the end of experiment (January; Fig. 5). However, no significant difference in gonadal aromatase activity between control males and AI males (1.4 ± 0.5 vs. 0.5 ± 0.3 pmole/h·mg protein, P > 0.05) was found (Fig. 5).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The timing of sex change and transformation in the gonad is still not known during the natural sex change process in this species. Ovarian tissue started to develop in both putative females and males during postspawning season in black porgy after 2 yr of age, and it became the dominant tissue in the bisexual gonad during the nonspawning season [3]. In the current experiments, the treatment of aromatase inhibitors had seasonal effects in the physiological changes of sex change. The gonadal stage was already different in June (after 3 mo of AI feeding) between AI and control groups. Testicular tissue in the AI group became more developed with higher 11-KT production in June–September and later, compared with the control group. The data suggest that in the natural sex change, the gonadal change probably starts to occur during the nonspawning season (May–August). June may be the critical time that gonadal transformation may occur during the natural sex change process. Our preliminary experiment failed to block natural sex change when aromatase inhibitors were given during July–October (unpublished data). Detailed experiments should be conducted to examine the time course related to sex change in black porgy. The difficulties of these experiments were to give the AIs to black porgy for a long time and also to keep the marine fish until the coming spawning (i.e., the third) season. The completion of natural sex change therefore could be judged on the basis of the criteria "collectable oocytes," "spermiation," or "gonad dissection" during the spawning season. We had 40% and 60% survival rates in the control and AI-fed groups, respectively, after 9 mo of treatment. No mortality due to sampling or feeding with AIs was observed in these experiments.
Our findings are also consistent with the current concept that aromatase is important for the female sex differentiation in gonochoristic species. Gonadal aromatase activity is related to ovarian development in fish and other animals [20–22]. The importance of aromatase in sex differentiation has been further shown by applying aromatase inhibitors. Masculinization of gonads by aromatase inhibitors has been obtained in several gonochoristic animals, included chinook salmon (Oncorhynchus tshawytscha) [9], rainbow trout (O. mykiss) [23], tilapia (Oreochromis niloticus) [23, 24], Japanese flounder (Paralichthys olivaceus) [24, 25], newts (P. waltl) [16], lizards (Cnemidophorus uniparens) [14, 26], slider turtles (Trachemys scripta) [12, 14] and snapping turtles (Chelydra serpentina) [13], and birds [15]. Fadrozole also further induced the sex change in a protogynous blackeye goby, Coryphopterus nicholsii [27].
AIs also enhanced male function in our studies, on the basis of the timing at which spermiation begins during the spawning season, the amount of milt volume in spermiation, and increased plasma 11-KT concentrations. Long-term oral administration of AIs resulted in the onset of spermiation at least 3 mo earlier and with a much larger volume of sperm as compared with the control group. Male functions were not pronounced (only small milt volume) in 3-yr-old males as shown in our control group. Plasma 11-KT concentrations were higher (2-fold to 4-fold) in the AI group than in the control group. 11-KT is the major androgen in many teleosts, and the concentrations of this steroid in plasma are correlated with the spermiation in black porgy [4, 5]. The stimulation of spermiation by AIs was further supported by a decreased milt volume after withdrawing the administration of AIs from some individuals in the AI-fed group (unpublished data). Withdrawal of AI in the AI-fed black porgy resulted in a decrease in milt production (it became 20%–32% in milt volume compared with the AI-fed males; unpublished data). Implantation of ATD also induced maturation in three-spined stickleback males (Gasterosteus aculeatus) [28]. However, ATD decreased spermiation and LH concentrations in Atlantic salmon (Salmo salar) [29]. The effects of AIs in fish reproduction may vary according to the effects of aromatase-dependent feedback effects on gonadotropins, species differences, and the stage of fish as presented in these experiments and other studies [28, 29]. The possible direct effects of AIs on gonadal function should not be excluded.
We also found that plasma LH levels were significantly higher in May and August–November in fish that were administered AIs than in controls. The profiles of plasma LH levels in the AI group were consistent with those for spermiation volume and plasma 11-KT levels except in June and July. The stimulation of plasma LH by AIs is possibly due to the aromatase-dependent negative feedback effect in LH of black porgy. Injection with AIs resulted in a different degree of stimulation in plasma LH levels in black porgy [18]. Injection of E2 (the product of aromatase) significantly stimulated LH release in black porgy with a seasonal change [8, 30]. The aromatase-dependent positive and negative regulation of steroids on FSH or LH but with a seasonal variation also had been indicated in Atlantic salmon [29].
Oral administration of AIs strongly inhibited aromatase activities in various regions of brain, pituitary, and gonad. These inhibitions possibly caused the suppression of estrogen production and the blockage of the natural sex change in black porgy. The order of aromatase activity in the gonad is as follows: ovary in females (control) > bisexual gonads in functional males (control) > good functional testes in males (AI-treated). Aromatase activity in AI-males was lower (but there was no statistical difference) than that in control males because AI-males had a functional testis as a gonad, and control males had a spermiating but bisexual gonad. The data also support that AI stimulated the development of male testis and milt production. At the end of the AI feeding (in January), in addition to the difference in aromatase activity and milt volume between AI males and control males/females, AI males (200.9 ± 4.3 pg/ml) also had higher 11-KT levels than control males (101.0 ± 34.0 pg/ml) or than females (32.5 ± 4.7 pg/ml), but no difference in plasma LH levels was found between AI (4.3 ± 0.8 ng/ml) and control males/females (4.2 ± 0.2 ng/ml).
Aromatase (mostly in gonad) and estrogen have been indicated to play a major role in the female development of animals described earlier in this discussion. However, other factors except gonadal aromatase should not be excluded for the mechanism of gonadal sex change in black porgy. Short-term injection with fadrozole and ATD also significantly inhibited aromatase activity in brain but not in gonad in our previous studies [18]. The suppression of aromatase activity in brain and pituitary probably also involved in the aromatase-dependent effects in the regulation of gonadotropin and induced high levels of plasma LH. It is interesting to point out the concept that high levels of plasma LH are associated with the development toward the male sex in this study. The increased plasma LH levels were probably not only beneficial for spermiation, but also for maintaining the male phase in the hermaphrodite fish. Our recent data in another experiment also showed that concentrations of plasma LH during the nonspawning season were higher in 3-yr-old males than following the natural sex change to females (unpublished data). Further experiments are needed to investigate this suggestion. The involvement of brain and pituitary aromatase activity in sex change is not known in animals. High aromatase activity in brain has been hypothesized to play a major role in female developmental pathway in turtles (T. scripta) [31]. Brain aromatase may be involved in the estrogen-dependent sexual behavior in male ring dove, Streptopelia risoria [32].
In conclusion, aromatase inhibitors consistently suppressed endogenous aromatase activity, blocked the natural sex change, and stimulated high levels of plasma LH and 11-KT. AIs also induced spermiation, a larger precocious spermiation volume, and probably inhibited the development of ovarian tissue. The present experiments clearly showed that aromatase plays an important role in the occurrence of the natural sex change in the protandrous black porgy.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported in part by the National Science Council of Taiwan, NSC 91-2313-B019-009. 
2 Correspondence: Ching-Fong Chang, Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan, Republic of China. FAX: 886 2 2462 1579; b0044{at}mail.ntou.edu.tw
Accepted: January 8, 2002.
Received: August 31, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus). Mol Reprod Dev 2000; 56:1-5[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  G.-C. Wu, S. Tomy, M. Nakamura, and C.-F. Chang Dual Roles of cyp19a1a in Gonadal Sex Differentiation and Development in the Protandrous Black Porgy, Acanthopagrus schlegeli Biol Reprod, December 1, 2008; 79(6): 1111 - 1120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G.-C. Wu, S. Tomy, and C.-F. Chang The Expression of nr0b1 and nr5a4 During Gonad Development and Sex Change in Protandrous Black Porgy Fish, Acanthopagrus schlegeli Biol Reprod, February 1, 2008; 78(2): 200 - 210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.-T. Wong, S. Ijiri, and Y. Zohar Molecular Biology of Ovarian Aromatase in Sex Reversal: Complementary DNA and 5'-Flanking Region Isolation and Differential Expression of Ovarian Aromatase in the Gilthead Seabream (Sparus aurata) Biol Reprod, May 1, 2006; 74(5): 857 - 864. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-H. Twan, J.-S. Hwang, Y.-H. Lee, S.-R. Jeng, W.-S. Yueh, Y.-H. Tung, H.-F. Wu, S. Dufour, and C.-F. Chang The Presence and Ancestral Role of Gonadotropin-Releasing Hormone in the Reproduction of Scleractinian Coral, Euphyllia ancora Endocrinology, January 1, 2006; 147(1): 397 - 406. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P Black, J. Balthazart, M. Baillien, and M. S Grober Socially induced and rapid increases in aggression are inversely related to brain aromatase activity in a sex-changing fish, Lythrypnus dalli Proc R Soc B, November 22, 2005; 272(1579): 2435 - 2440. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-H. Lee, G.-C. Wu, J.-L. Du, and C.-F. Chang Estradiol-17{beta} Induced a Reversible Sex Change in the Fingerlings of Protandrous Black Porgy, Acanthopagrus schlegeli Bleeker: The Possible Roles of Luteinizing Hormone in Sex Change Biol Reprod, October 1, 2004; 71(4): 1270 - 1278. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
