Yan-Horn Lee

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.

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.

Purification of the sex steroid-binding protein from common carp (Cyprinus carpio) plasma

1. Sex steroid-binding protein was purified from common carp plasma. 2. Testosterone- and estradiol-binding activity existed at the same fraction eluted from gel Sepharose CL-2B, DEAE-Sephacel, hydroxylapatite and HPLC. 3. The molecular weight of the sex steroid-binding protein was 194,000. 4. At 50% displacement the order in which the steroids displaced [3H]testosterone bound to the binding protein was as follows: androstenedione greater than estradiol-17 beta greater than 11-deoxy-17-hydroxycorticosterone greater than 17 alpha-hydroxyprogesterone greater than progesterone greater than deoxycorticosterone greater than estrone greater than 11-ketotestosterone greater than 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one greater than androstenedione greater than pregnenolone greater than cortisone greater than cortisol.

Yolk formation in a stony coral Euphyllia ancora (Cnidaria, Anthozoa): insight into the evolution of vitellogenesis in nonbilaterian animals.

Vitellogenin (Vg) is a major yolk protein precursor in numerous oviparous animals. Numerous studies in bilateral oviparous animals have shown that Vg sequences are conserved across taxa and that Vgs are synthesized by somatic-cell lineages, transported to and accumulated in oocytes, and eventually used for supporting embryogenesis. In nonbilateral animals (Polifera, Cnidaria, and Ctenophora), which are regarded as evolutionarily primitive, although Vg cDNA has been identified in 2 coral species from Cnidaria, relatively little is known about the characteristics of yolk formation in their bodies. To address this issue, we identified and characterized 2 cDNA encoding yolk proteins, Vg and egg protein (Ep), in the stony coral Euphyllia ancora. RT-PCR analysis revealed that expression levels of both Vg and Ep increased in the female colonies as coral approached the spawning season. In addition, high levels of both Vg and Ep transcripts were detected in the putative ovarian tissue, as determined by tissue distribution analysis. Further analyses using mRNA in situ hybridization and immunohistochemistry determined that, within the putative ovarian tissue, these yolk proteins are synthesized in the mesenterial somatic cells but not in oocytes themselves. Furthermore, Vg proteins that accumulated in eggs were most likely consumed during the coral embryonic development, as assessed by immunoblotting. The characteristics of Vg that we identified in corals were somewhat similar to those of Vg in bilaterian oviparous animals, raising the hypothesis that such characteristics were likely present in the oogenesis of some common ancestor prior to divergence of the cnidarian and bilaterian lineages.

Germ cell development in the scleractinian coral Euphyllia ancora (Cnidaria, Anthozoa).

Sexual reproduction of scleractinian coral is among the most important means of establishing coral populations. However, thus far, little is known about the mechanisms underlying coral gametogenesis. To better understand coral germ cell development, we performed a histological analysis of gametogenesis in Euphyllia ancora and characterized the coral homolog of the Drosophila germline marker gene vasa. The histological analysis revealed that E. ancora gametogenesis occurs in the mesenterial mesoglea between the mesenterial filaments and the retractor muscle bands. The development of germ cells takes approximately one year in females and half a year in males. Staining of tissue sections with an antibody against E. ancora Vasa (Eavas) revealed anti-Eavas immunoreactivity in the oogonia, early oocyte, and developing oocyte, but only faint or undetectable reactivity in developing oocytes that were >150 µm in diameters. In males, Eavas could be detected in the spermatogonia and primary spermatocytes but was only faintly detectable in the secondary spermatocytes, spermatids, and sperms. Furthermore, a reverse transcription-polymerase chain reaction analysis and Western blotting analysis of unfertilized mature eggs proved the presence of Eavas transcripts and proteins, suggesting that Eavas may be a maternal factor. Vasa may represent a germ cell marker for corals, and would allow us to distinguish germ cells from somatic cells in coral bodies that have no distinct organs.

17β-Estradiol, but not testosterone stimulates gonadotropin II concentrations in the protandrous black porgy, Acanthopagrus schlegeli Bleeker

The objective of the present study was to investigate the in vivo effects of different doses of 17β-estradiol (E2) and testosterone (T) on the levels of plasma and pituitary gonadotropin II (GTH II) in 2-year-old black porgy, Acanthopagrus schlegeli, during the spawning season. Male fish were distributed among 7xa0groups (nxa0=xa049), control, E2or T (with 3xa0doses, 2.4xa0ng, 72xa0ng and 2.2xa0μgxa0g−1 body weight). Fish were injected with the respective vehicle or different doses of E2 or T on days 1 and 14. Plasma E2 levels were significantly increased in the 72xa0ng E2 group on daysxa08 and 14. Plasma vitellogenin levels were significantly higher in the 72xa0ng E2 group on daysxa014 and 20, and 2.2xa0μg E2 group on daysxa08, 14 and 20 than those in the control group. Plasma GTH II concentrations were significantly higher in the 2.2xa0μg E2 group than in the control and other E2groups on days 8, 14 and 20. Pituitary GTHxa0II contents was significantly higher in the 7.2xa0ng E2 group compared to the control and other E2groups on day 20. Plasma GTH II concentrations were similar in the control and all the T groups on daysxa08, 14 and 20. None of the doses of T treatment stimulated pituitary GTHxa0II content on dayxa020, although plasma vitellogenin levels were elevated. It is concluded that GTHxa0II synthesis and secretion in black porgy is stimulated by an estrogen-specific effect.

Characterization of the plasma sex steroid-binding protein in eel (Anguilla japonica)

Abstract Sex steroid-binding protein was purified from the plasma of an eel, Anguilla japonica . Testosterone and estradiol-binding activity existed at the same fraction when eluted from gel Sepharose CL-2B, DEAE-Sephacel, hydroxylapatite and HPLC. The mass weight of the sex steroid-binding protein was 64,000 Da. At 50% displacement, in which the steroids displaced tritiated testosterone or estradiol-17β bound to the binding protein, the effect was as follows: estradiol-17β, androstenediol and testosterone were the most effective competitors, 11-ketotestosterone competed less well, and another 10 steroids were poor competitors. The data suggest that the structure with specific C- and D-rings and hydroxyl groups on carbon 17 of the steroids is important for the strong interaction of steroid and sex steroid-binding protein in the eel.

A Novel Female-Specific and Sexual Reproduction-Associated Dmrt Gene Discovered in the Stony Coral, Euphyllia ancora.

ABSTRACT Transcription factors encoded by the Dmrt gene family regulate multiple aspects of animal reproduction. Most studies investigating the Dmrt gene family were conducted in model organisms from bilateral species, with a particular emphasis on gene function in male sex determination. It is still unclear whether the E. ancora Dmrt (EaDmrt) genes found in basal metazoans such as cnidarians share similar characteristics with orthologs in other metazoans. In this study, seven full Dmrt gene transcript sequences for a gonochoric coral, Euphyllia ancora (phylum: Cnidaria; class: Anthozoa), were obtained through transcriptome data mining, RT-PCR analysis, rapid amplification of cDNA ends, and sequencing. These EaDmrts were subjected to quantitative assays measuring temporal and tissue-specific expression. Results demonstrated a unique gene expression pattern for EaDmrtE, which is enriched in female germ cells during the spawning season. Based on the phylogenetic analyses performed across the homologous Dmrt genes in metazoans, we found that the female-specific EaDmrtE gene is not related to the DM1 gene of Acropora spp. coral nor to Dmrt1 of vertebrates, which are involved in sexual reproduction, especially in sex determination (vertebrate Dmrt1). Additionally, high levels of EaDmrtE transcripts detected in unfertilized mature eggs are retained in newly formed zygotes but decrease during embryonic development. We suggest that the newly discovered gene may play a role in oogenesis and early embryogenesis as a maternal factor in corals. Therefore, the sexual reproduction-associated Dmrt gene(s) should have arisen in cnidarians and might have evolved multiple times in metazoans.