Jin-Lien Du

Aromatase Inhibitors Block Natural Sex Change and Induce Male Function in the Protandrous Black Porgy, Acanthopagrus schlegeli Bleeker: Possible Mechanism of Natural Sex Change

Abstract The objectives of the present study were to investigate the effects of oral administration of aromatase inhibitors on sex change, milt volume, 11-ketotestosterone (11-KT), and LH in plasma; aromatase activity in gonad, pituitary, and brain in the protandrous fish, black porgy (Acanthopagus schlegeli Bleeker). Two-year-old functional male black porgy were divided into two groups; one was fed a control diet and the other was fed a diet mixed with aromatase inhibitors (AIs; fadrozole and 1,4,6-androstatriene-3,17-dione, each 10 mg/kg feed) for 8.5 mo. A significantly higher gonadosomatic index was observed in the AI group. Fish treated with AIs showed complete suppression of natural sex change. Significantly higher levels of plasma 11-KT, LH, and milt volume were shown in the AI group than the controls. Lower aromatase activity in the gonad, pituitary, forebrain, midbrain, and hindbrain in concordance with the suppression of sex change was observed in the AI group. The data show that aromatase is directly involved in the mechanism of natural sex change of protandrous black porgy. AIs also enhanced male function in concordance with the elevated plasma levels of 11-KT and spermiation in milt volume.

Differential Dmrt1 transcripts in gonads of the protandrous black porgy, Acanthopagrus schlegeli

Black porgy, Acanthopagrus schlegeli Bleeker, is a marine protandrous hermaphrodite fish. A Dmrt1 cDNA was cloned and characterized and in order to study the process of sex change in this species, mRNA transcripts of Dmrt1 were monitored. Dmrt1 was specifically transcribed in testis and seminal vesicle in 2-year-old black porgy according to RT-PCR and Southern analysis. A real-time quantification PCR analysis was further developed for the measurement of Dmrt1 transcripts. Dmrt1 transcripts were at significantly higher levels in bisexual testis than bisexual ovary in 1+ and 2+ year-old fish. Dmrt1 transcripts decreased in the functional and bisexual testis of 3-year-old fish. Much higher levels of Dmrt1 transcripts in the bisexual ovary were detected in 1+ year-old fish than in 2+ and 3-year-old fish. No differences in Dmrt1 transcripts were found in bisexual ovaries of 2+ and 3-year-old fish and female ovaries of 3-year-old fish. The data suggest there is relationship of Dmrt1 to the sex change of protandrous black porgy.

Estradiol-17β Induced a Reversible Sex Change in the Fingerlings of Protandrous Black Porgy, Acanthopagrus schlegeli Bleeker: The Possible Roles of Luteinizing Hormone in Sex Change

Abstract The objectives of the present study were to investigate the effects of oral administration of estradiol-17β (E2) on luteinizing hormone (LH) in plasma, aromatase activity in gonad, and sex change in the fingerlings of protandrous black porgy, Acanthopagus schlegeli Bleeker. The expression of estrogen receptor (ER) and androgen receptor (AR) transcripts in gonad was also analyzed. Undifferentiated (2-mo-old) black porgy were divided into two groups, one fed a control diet and the other a diet mixed with E2 (6.0 mg/kg feed) for 5 mo. Fish treated with E2 for 3 mo showed complete suppression of spermatogenesis and spermiation and induced sex change with primary oocytes. Aromatase activity in forebrain and midbrain was increased in the control in December–March (during the spawning season). E2 stimulated aromatase activity in the brain. Higher gonadal aromatase activity in concordance with elevated levels of plasma LH was observed in the E2 group compared with the control. After 2-mo of E2 termination, regressed testicular tissue recovered and controlled females gradually reversed back to functional males in January and March. Plasma LH levels were higher in the E2-terminated group during the period of reversible sex change (from a controlled female to male) compared with the control. The expression of ER and AR transcripts was closely related to the development of testis and ovary. The data showed that E2 induced a reversible sex change with high plasma LH. Increase of gonadal aromatase and decrease of ER/AR were associated with controlled sex change. Plasma LH levels were correlated with the conversion from a controlled female to male in black porgy.

Regulation of plasma gonadotropin II secretion by sex steroids, aromatase inhibitors, and antiestrogens in the protandrous black porgy, Acanthopagrus schlegeli Bleeker.

Plasma gonadotropin II (GTH II) concentrations were significantly higher (approx. 15-20-fold) in estradiol-17beta (E(2)) treated (1.0 microg or 2.5 microg g(-1) body weight) female black porgy from days 4 to 12 compared with the control. E(2) (1 microg g(-1) wt.) had a stronger stimulation on plasma GTH II in early recrudescent phase (low GSI) males (11-fold) than in high GSI and late spermiating males (2.6-fold, P< 0.05). No effect of androgens (testosterone, T; 5alpha-dihydrotestosterone, DHT) on plasma GTH II levels was observed either sex. The levels of plasma GTH II were stimulated in 1,4,6-androstatriene-3,17-dione (ATD, 1 microg g(-1), 2 microg g(-1) body wt.) and fadrozole-treated (1 microg g(-1), 3 microg g(-1) body wt.) groups compared to control. Tamoxifen (1 microg g(-1), 3 microg g(-1) body wt.) but not enclomiphene could stimulate high GTH II levels in plasma. In another experiment of ATD in combination with T, T treatment further attenuated the ATD stimulation of plasma GTH II levels. We concluded that GTH II secretion is positively regulated by an estrogen-specific effect in female and male black porgy. Gonadal stage had significant effects on the responsiveness of GTH II to E(2) stimulation in males. A negative aromatase-dependent feedback control of plasma GTH II levels was also suggested in the protandrous black porgy, Acanthopagrus schlegeli.

Current status of genetic and endocrine factors in the sex change of protandrous black porgy, Acanthopagrus schlegeli (Teleostean).

Abstract: Black porgy, Acanthopagrus schlegeli Bleeker, a marine protandrous hermaphrodite fish, is functionally male for the first 2 years of life, but begins to sexually change to female after the third year. Testicular tissue and ovarian tissue are separated by connective tissue in the bisexual gonad. This sex pattern provides a unique model to study the mechanism of sex change in fish. The annual profiles of plasma estradiol, vitellogenin, and 11‐ketotestosterone concentrations in males were significantly different from those in the 3‐year‐old females. Oral administration of estradiol stimulated high levels of gonadal aromatase activity, plasma luteinizing hormone (LH) levels, and sex change in the 2‐year‐old fish. Oral administration with aromatase inhibitors for 1 year further blocked the natural sex change in 3‐year‐old black porgy and all fish became functional males. Transcripts of estrogen receptor (ER), androgen receptor, and gonadotropin receptors in the ovarian tissue of bisexual gonad were significantly less expressed than those in the bisexual testicular tissue. ER and aromatase transcripts were much higher in the vitellogenic ovary than those in the bisexual ovarian tissue. Plasma LH levels were higher in male fish than sex‐changing fish during postspawning and nonspawning season in 2+‐year‐old black porgy. We are also conducting investigations on the role of the genetic factors (Dmrt 1, Sox 9, Sf‐1, and Dax‐1) in sex development and sex change. An endocrine mechanism of sex change in black porgy is proposed.

Differential Messenger RNA Transcription of Androgen Receptor and Estrogen Receptor in Gonad in Relation to the Sex Change in Protandrous Black Porgy, Acanthopagrus schlegeli

Abstract Black porgy, Acanthopagrus schlegeli Bleeker, is a marine protandrous hermaphrodite fish. All are functional males at 1–2 yr of age and then become either males or females at 3 yr of age. To study the process of sex change in this species, mRNA transcripts of two estrogen receptors (ERα and ERβ) and an androgen receptor (AR) were monitored. An AR cDNA was cloned and characterized. ERα, ERβ, and AR were differentially transcribed in bisexual testicular and ovarian tissue according to reverse transcription polymerase chain reaction (RT-PCR) and Southern analysis. A real-time quantification PCR analysis was further developed for the measurement of AR, ERα, and ERβ transcripts. ERα and AR transcripts were significantly more plentiful in bisexual testis than in bisexual ovary in 1+- and 2+-yr-old fish. ERα, ERβ, and AR transcripts decreased in the functional testis of 3-yr-old fish. Similar levels of ERβ and AR were detected in the ovary of sex-changed females and in functional testis of 3-yr-old males. Significantly decreased AR transcripts were found in testicular tissue of bisexual and functional male and female gonads in 3-yr-old fish as compared with 1- and 2-yr-old fish. In contrast, increased ERα transcripts were detected in the bisexual ovary and sex-changed ovary of 3-yr-old fish as compared with the bisexual ovary of 1- and 2-yr-old fish. The data suggest a differential sensitivity in the bisexual testicular and ovarian tissue of black porgy.

Seasonal Profiles of Brain and Pituitary Gonadotropin-Releasing Hormone and Plasma Luteinizing Hormone in Relation to Sex Change of Protandrous Black Porgy, Acanthopagrus schlegeli

Abstract Three molecular variants of GnRH in the brain (sbGnRH, sGnRH, and cGnRH-II) and two forms in the pituitary (sbGnRH and sGnRH) were detected in protandrous black porgy, Acanthopagrus schlegeli using chromatographic and immunological methods. In juvenile fish, brain sbGnRH, sGnRH, and cGnRH-II levels increased in May and reached their highest levels in July and August (the nonspawning season) and in January through March (the spawning season). In fish aged 1 yr and older, high levels of brain sbGnRH and sGnRH were detected in September, November, and February–March, but the levels of brain cGnRH-II remained constant. A gradual increase in pituitary sbGnRH was detected in juvenile fish from July to March. In fish aged 1+ yr, pituitary sbGnRH levels were high in September and March–May, but low in January–February. A close correlation between pituitary sbGnRH and plasma LH levels was found in juvenile fish and in those aged 1+ yr. In fish aged 2+ yr, significantly lower levels of plasma LH was detected during the nonspawning period in fish that changed sex compared with the fish that remained as males. Higher plasma LH levels were detected in the sex-changing fish from artificially sex-reversed female to male. FSH receptor and LH receptor transcripts were higher in bisexual testicular tissue than in ovarian tissue in 2+-yr-old fish. Direct effects of hCG on sex change were studied and the results show that exogenous hCG did not stimulate gonadal aromatase activity in 2+-yr-old fish. Therefore, it is suggested that high and basal levels of plasma LH during the nonspawning season correlate with the development of male and female gonad, respectively, in black porgy. This important role of the neuroendocrine system in sex change (for male direction) is proposed in hermaphroditic fish.